U.S. patent application number 15/552892 was filed with the patent office on 2018-02-15 for cellobiohydrolase variants and polynucleotides encoding same.
This patent application is currently assigned to Novozymes A/S. The applicant listed for this patent is Novozymes A/S. Invention is credited to Silke Flindt Badino, Kim Borch, Brett McBrayer, Peter Westh, Michael Skovbo Windahl.
Application Number | 20180044652 15/552892 |
Document ID | / |
Family ID | 55487165 |
Filed Date | 2018-02-15 |
United States Patent
Application |
20180044652 |
Kind Code |
A1 |
McBrayer; Brett ; et
al. |
February 15, 2018 |
Cellobiohydrolase Variants and Polynucleotides Encoding Same
Abstract
The present invention relates to cellobiohydrolase variants. The
present invention also relates to polynucleotides encoding the
variants; nucleic acid constructs, vectors, and host cells
comprising the polynucleotides; and methods of using the
variants.
Inventors: |
McBrayer; Brett;
(Sacramento, CA) ; Windahl; Michael Skovbo;
(Stenlose, DK) ; Westh; Peter; (Copenhagen,
DK) ; Badino; Silke Flindt; (Bagsvaerd, DK) ;
Borch; Kim; (Birkerod, DK) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Novozymes A/S |
Bagsvaerd |
|
DK |
|
|
Assignee: |
Novozymes A/S
Bagsvaerd
DK
|
Family ID: |
55487165 |
Appl. No.: |
15/552892 |
Filed: |
February 24, 2016 |
PCT Filed: |
February 24, 2016 |
PCT NO: |
PCT/US2016/019404 |
371 Date: |
August 23, 2017 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
62120178 |
Feb 24, 2015 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C13K 1/02 20130101; C12Y
302/01 20130101; C12Y 302/01176 20130101; C12N 9/2437 20130101;
C12Y 302/01091 20130101; C12P 19/02 20130101 |
International
Class: |
C12N 9/42 20060101
C12N009/42; C12P 19/02 20060101 C12P019/02; C13K 1/02 20060101
C13K001/02 |
Claims
1. A cellobiohydrolase variant, comprising an alteration at one or
more positions corresponding to positions 4, 44, 45, 72, 265, 266,
391, 393 and 394 of SEQ ID NO: 4, wherein the variant has
cellobiohydrolase activity.
2. The cellobiohydrolase variant of claim 1, wherein the variant
has at least 60%, e.g., at least 65%, at least 70%, at least 75%,
at least 80%, at least 81%, at least 82%, at least 83%, at least
84%, at least 85%, at least 86%, at least 87%, at least 88%, at
least 89%, at least 90%, at least 95%, at least 96%, at least 97%,
at least 98%, or at least 99%, but less than 100%, sequence
identity to the mature polypeptide of a parent
cellobiohydrolase.
3. The cellobiohydrolase variant of claim 1, wherein the alteration
is a substitution.
4. The cellobiohydrolase variant of claim 1, wherein the parent
cellobiohydrolase is selected from the group consisting of: (a) a
polypeptide having at least 60% sequence identity to the mature
polypeptide of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO:
10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ
ID NO: 20, SEQ ID NO: 22 or SEQ ID NO: 52; (b) a polypeptide
encoded by a polynucleotide that hybridizes under low stringency
conditions with (i) the mature polypeptide coding sequence of SEQ
ID NO: 1, SEQ ID NO: 5, ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ
ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:
21 or SEQ ID NO: 51, (ii) the cDNA sequence thereof, or (iii) the
full-length complement of (i) or (ii); (c) a polypeptide encoded by
a polynucleotide having at least 60% identity to the mature
polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID
NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15,
SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 51, or the
cDNA sequence thereof; and (d) a fragment of the mature polypeptide
of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID
NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20,
SEQ ID NO: 22, or SEQ ID NO: 52 which has cellobiohydrolase
activity.
5. The cellobiohydrolase variant of claim 1, wherein the parent
cellobiohydrolase has at least 60%, at least 65%, at least 70%, at
least 75%, at least 80%, at least 81%, at least 82%, at least 83%,
at least 84%, at least 85%, at least 86%, at least 87%, at least
88%, at least 89%, at least 90%, at least 95%, at least 96%, at
least 97%, at least 98%, at least 99%, or 100% sequence identity to
the mature polypeptide of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8,
SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID
NO: 18, SEQ ID NO: 20, SEQ ID NO: 22 or SEQ ID NO: 52.
6. The cellobiohydrolase variant of claim 1, which comprises one of
more substitutions selected from: a substitution at a position
corresponding to position 4 of SEQ ID NO: 4, wherein the
substitution is with Cys; a position corresponding to position 44
of SEQ ID NO: 4, wherein the substitution is with Ser, Thr, Ala,
Gly, Ile, Met, Asn, or Lys; a substitution at a position
corresponding to position 45 of SEQ ID NO: 4, wherein the
substitution is with Ser or Asn; a substitution at a position
corresponding to position 72 of SEQ ID NO: 4, wherein the
substitution is with Cys; a substitution at a position
corresponding to position 265 of SEQ ID NO: 4, wherein the
substitution is with Gly, Pro, or Ala; a substitution at a position
corresponding to position 266 of SEQ ID NO: 4, wherein the
substitution is with Tyr; a substitution at a position
corresponding to position 391 of SEQ ID NO: 4, wherein the
substitution is with Asp, Trp, Asn, or Val; a substitution at a
position corresponding to position 393 of SEQ ID NO: 4, wherein the
substitution is with Asp; and a substitution at a position
corresponding to position 394 of SEQ ID NO: 4, wherein the
substitution is with Pro.
7. The cellobiohydrolase variant of claim 1, which comprises or
consists of SEQ ID NO: 26 or the mature polypeptide thereof (e.g.,
amino acids 1-506 of SEQ ID NO: 26), SEQ ID NO: 28 or the mature
polypeptide thereof (e.g., amino acids 1-506 of SEQ ID NO: 28), SEQ
ID NO: 38 or the mature polypeptide thereof (e.g., amino acids
1-506 of SEQ ID NO: 38), SEQ ID NO: 42 or the mature polypeptide
thereof (e.g., amino acids 1-506 of SEQ ID NO: 42), SEQ ID NO: 46
or the mature polypeptide thereof (e.g., amino acids 1-506 of SEQ
ID NO: 46), SEQ ID NO: 48 or the mature polypeptide thereof (e.g.,
amino acids 1-506 of SEQ ID NO: 48), SEQ ID NO: 63 or the mature
polypeptide thereof (e.g., amino acids 1-503 of SEQ ID NO: 63), SEQ
ID NO: 67 or the mature polypeptide thereof (e.g., amino acids
1-503 of SEQ ID NO: 67), SEQ ID NO: 69 or the mature polypeptide
thereof (e.g., amino acids 1-503 of SEQ ID NO: 69), SEQ ID NO: 71
or the mature polypeptide thereof (e.g., amino acids 1-503 of SEQ
ID NO: 71), SEQ ID NO: 73 or the mature polypeptide thereof (e.g.,
amino acids 1-503 of SEQ ID NO: 73), SEQ ID NO: 75 or the mature
polypeptide thereof (e.g., amino acids 1-503 of SEQ ID NO: 75), SEQ
ID NO: 77 or the mature polypeptide thereof (e.g., amino acids
1-503 of SEQ ID NO: 77), SEQ ID NO: 79 or the mature polypeptide
thereof (e.g., amino acids 1-503 of SEQ ID NO: 79), SEQ ID NO: 81
or the mature polypeptide thereof (e.g., amino acids 1-503 of SEQ
ID NO: 81), SEQ ID NO: 83 or the mature polypeptide thereof (e.g.,
amino acids 1-503 of SEQ ID NO: 83), SEQ ID NO: 85 or the mature
polypeptide thereof (e.g., amino acids 1-503 of SEQ ID NO: 85).
8. The cellobiohydrolase variant of claim 1, wherein the parent is
a fusion protein in which a heterologous carbohydrate binding
domain is fused to the parent.
9. A cellobiohydrolase variant, comprising an alteration at one or
more positions corresponding to positions 4, 44, 45, 72, 265, 266,
391, 393 and 394 of SEQ ID NO: 4, wherein the variant has
cellobiohydrolase activity, and wherein the variant comprises a
catalytic domain having at least 60%, e.g., at least 65%, at least
70%, at least 75%, at least 80%, at least 81%, at least 82%, at
least 83%, at least 84%, at least 85%, at least 86%, at least 87%,
at least 88%, at least 89%, at least 90%, at least 95%, at least
96%, at least 97%, at least 98%, or at least 99%, but less than
100%, sequence identity to the catalytic domain of a parent
cellobiohydrolase.
10. The cellobiohydrolase variant of claim 9, wherein the parent
cellobiohydrolase comprises a catalytic domain having at least 60%,
at least 65%, at least 70%, at least 75%, at least 80%, at least
81%, at least 82%, at least 83%, at least 84%, at least 85%, at
least 86%, at least 87%, at least 88%, at least 89%, at least 90%,
at least 95%, at least 96%, at least 97%, at least 98%, at least
99%, or 100% sequence identity to the amino acids 1 to 437 of SEQ
ID NO: 4, amino acids 1 to 429 of SEQ ID NO: 6, amino acids 1 to
440 of SEQ ID NO: 8, amino acids 1 to 437 of SEQ ID NO: 10, amino
acids 1 to 437 of SEQ ID NO: 12, amino acids 1 to 437 of SEQ ID NO:
14, amino acids 1 to 437 of SEQ ID NO: 16, amino acids 1 to 430 of
SEQ ID NO: 18, amino acids 1 to 433 of SEQ ID NO: 20, amino acids 1
to 438 of SEQ ID NO: 22, or amino acids 1 to 437 of SEQ ID NO:
52.
11. An isolated polynucleotide encoding the cellobiohydrolase
variant of claim 1.
12. A host cell comprising the polynucleotide of claim 11.
13. A method of producing a cellobiohydrolase variant, comprising:
(a) cultivating the host cell of claim 12 under conditions suitable
for expression of the variant; and (b) recovering the variant.
14. A transgenic plant, plant part or plant cell transformed with
the polynucleotide of claim 11.
15. A method of producing the variant of claim 1, comprising: (a)
cultivating a transgenic plant or a plant cell comprising a
polynucleotide encoding the variant under conditions conducive for
production of the variant; and (b) recovering the variant.
16. A method for obtaining a cellobiohydrolase variant, comprising:
(a) introducing into a parent cellobiohydrolase an alteration at
one or more positions corresponding to positions 4, 44, 45, 72,
265, 266, 391, 393 and 394 of SEQ ID NO: 4, wherein the variant has
cellobiohydrolase activity; and (b) recovering the variant.
17. A whole broth formulation or cell culture composition
comprising the cellobiohydrolase variant of claim 1.
18. A process for degrading a cellulosic material, comprising: (a)
treating the cellulosic material with an enzyme composition in the
presence of the cellobiohydrolase variant of claim 1; and (b)
recovering the degraded cellulosic material.
19. (canceled)
20. The process of claim 18, wherein the enzyme composition
comprises one or more enzymes selected from the group consisting of
a cellulase, a GH61 polypeptide having cellulolytic enhancing
activity, a hemicellulase, a catalase, an esterase, an expansin, a
laccase, a ligninolytic enzyme, a pectinase, a peroxidase, a
protease, and a swollenin.
21. A process for producing a fermentation product, comprising: (a)
saccharifying a cellulosic material with an enzyme composition in
the presence of the cellobiohydrolase variant of claim 1; (b)
fermenting the saccharified cellulosic material with one or more
fermenting microorganisms to produce the fermentation product; and
(c) recovering the fermentation product from the fermentation.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority benefit of U.S. provisional
application Ser. No. 62/120,178, filed on Feb. 24, 2015. The
content of this application is fully incorporated herein by
reference.
REFERENCE TO A SEQUENCE LISTING
[0002] This application contains a Sequence Listing in computer
readable form, which is incorporated herein by reference.
BACKGROUND OF THE INVENTION
Field of the Invention
[0003] The present invention relates to cellobiohydrolase variants,
polynucleotides encoding the variants, and methods of producing and
using the variants.
Description of the Related Art
[0004] Cellulose is a polymer of the simple sugar glucose
covalently linked by beta-1,4-bonds. Many microorganisms produce
enzymes that hydrolyze beta-linked glucans. These enzymes include
endoglucanases, cellobiohydrolases, and beta-glucosidases.
Endoglucanases digest the cellulose polymer at random locations,
opening it to attack by cellobiohydrolases. Cellobiohydrolases
sequentially release molecules of cellobiose from the ends of the
cellulose polymer. Cellobiose is a water-soluble beta-1,4-linked
dimer of glucose. Beta-glucosidases hydrolyze cellobiose to
glucose.
[0005] The conversion of lignocellulosic feedstocks into ethanol
has the advantages of the ready availability of large amounts of
feedstock, the desirability of avoiding burning or land filling the
materials, and the cleanliness of the ethanol fuel. Wood,
agricultural residues, herbaceous crops, and municipal solid wastes
have been considered as feedstocks for ethanol production. These
materials primarily consist of cellulose, hemicellulose, and
lignin. Once the lignocellulose is converted to fermentable sugars,
e.g., glucose, the fermentable sugars can easily be fermented by
yeast into ethanol.
[0006] WO 2011/050037 discloses Thielavia terrestris
cellobiohydrolase variants with improved thermostability. WO
2011/050037 discloses Aspergillus fumigatus cellobiohydrolase
variants with improved thermostability. WO 2005/028636 discloses
variants of Hypocrea jecorina Cel7A cellobiohydrolase I. WO
2005/001065 discloses variants of Humicola grisea Cel7A
cellobiohydrolase I, Hypocrea jecorina cellobiohydrolase I, and
Scytalidium thermophilium cellobiohydrolase I. WO 2004/016760
discloses variants of Hypocrea jecorina Cel7A cellobiohydrolase I.
U.S. Pat. No. 7,375,197 discloses variants of Trichoderma reesei
cellobiohydrolase I.
[0007] Hybrid polypeptides comprising a cellobiohydrolase catalytic
domain and a carbohydrate binding module are described in e.g., WO
2010/060056, WO 2011/098551, WO 2011/153276, WO 2013/091577, and WO
2014/138672.
[0008] There is a need in the art for cellobiohydrolase variants
with improved properties to increase the efficiency of the
saccharification of lignocellulosic feedstocks.
[0009] The present invention provides cellobiohydrolase variants
with increased specific performance, polynucleotides encoding the
variants, and methods of producing and using the variants.
SUMMARY OF THE INVENTION
[0010] The present invention relates to isolated cellobiohydrolase
variants, comprising an alteration at one or more (e.g., several)
positions corresponding to positions 4, 44, 45, 72, 265, 266, 391,
393 and 394 of SEQ ID NO: 4, and wherein the variants have
cellobiohydrolase activity.
[0011] The present invention also relates to isolated
polynucleotides encoding the variants; nucleic acid constructs,
vectors, and host cells comprising the polynucleotides; and methods
of producing the variants.
[0012] The present invention also relates to processes for
degrading a cellulosic material, comprising: treating the
cellulosic material with an enzyme composition in the presence of a
cellobiohydrolase variant of the present invention. In one aspect,
the processes further comprise recovering the degraded cellulosic
material.
[0013] The present invention also relates to processes of producing
a fermentation product, comprising: (a) saccharifying a cellulosic
material with an enzyme composition in the presence of a
cellobiohydrolase variant of the present invention; (b) fermenting
the saccharified cellulosic material with one or more (e.g.,
several) fermenting microorganisms to produce the fermentation
product; and (c) recovering the fermentation product from the
fermentation.
[0014] The present invention also relates to processes of
fermenting a cellulosic material, comprising: fermenting the
cellulosic material with one or more (e.g., several) fermenting
microorganisms, wherein the cellulosic material is saccharified
with an enzyme composition in the presence of a cellobiohydrolase
variant of the present invention. In one aspect, the fermenting of
the cellulosic material produces a fermentation product. In another
aspect, the processes further comprise recovering the fermentation
product from the fermentation.
BRIEF DESCRIPTION OF THE FIGURES
[0015] FIG. 1 shows hydrolysis of microcrystalline cellulose by A.
fumigatus wild-type cellobiohydrolase I and the AC1-621 variant in
the presence of .beta.-glucosidase from A. fumigatus at 50.degree.
C. Values are shown in mM released glucose after 24 hours at pH
5.
[0016] FIG. 2 shows hydrolysis of microcrystalline cellulose by A.
fumigatus wild-type cellobiohydrolase I and the AC1-621 variant in
the presence of .beta.-glucosidase from A. fumigatus at 60.degree.
C. Values are shown in mM released glucose after 24 hours at pH
5.
[0017] FIG. 3 shows the effect of the Aspergillus fumigatus
cellobiohydrolase I AC1-621 variant and the Aspergillus fumigatus
cellobiohydrolase I AC1-596 wild-type parent on hydrolysis of
milled unwashed pretreated corn stover (PCS) by a cellulolytic
enzyme composition at 72 hours.
[0018] FIG. 4 shows the effect of the Aspergillus fumigatus
cellobiohydrolase I AC1-621 variant, the Aspergillus fumigatus
cellobiohydrolase I AC1-625 variant, and the Aspergillus fumigatus
cellobiohydrolase I AC1-596 wild-type parent on hydrolysis of
milled unwashed pretreated corn stover (PCS) by a cellulolytic
enzyme composition at 35.degree. C.
[0019] FIG. 5 shows the effect of the Aspergillus fumigatus
cellobiohydrolase I AC1-621 variant, the Aspergillus fumigatus
cellobiohydrolase I AC1-625 variant, and the Aspergillus fumigatus
cellobiohydrolase I AC1-596 wild-type parent on hydrolysis of
milled unwashed pretreated corn stover (PCS) by a cellulolytic
enzyme composition at 50.degree. C.
[0020] FIG. 6 shows the effect of the Aspergillus fumigatus
cellobiohydrolase I AC1-621 variant, the Aspergillus fumigatus
cellobiohydrolase I AC1-625 variant, and the Aspergillus fumigatus
cellobiohydrolase I AC1-596 wild-type parent on hydrolysis of
milled unwashed pretreated corn stover (PCS) by a cellulolytic
enzyme composition at 60.degree. C.
[0021] FIG. 7 shows the effect of the Aspergillus fumigatus
cellobiohydrolase I variants AC1-621, AC1-625, AC1-859, AC1-860,
AC1-870, AC1-939, AC1-940 and AC1-942, and the Aspergillus
fumigatus cellobiohydrolase I AC1-596 wild-type parent on
hydrolysis of milled unwashed pretreated corn stover (PCS) by a
cellulolytic enzyme composition at 50.degree. C., 55.degree. C. and
60.degree. C.
[0022] FIG. 8 shows the effect of the Aspergillus fumigatus
cellobiohydrolase I variant AC1-714, and the Aspergillus fumigatus
cellobiohydrolase I AC1-596 wild-type parent on hydrolysis of
milled unwashed pretreated corn stover (PCS) by a cellulolytic
enzyme composition at 50.degree. C., 55.degree. C. and 60.degree.
C.
[0023] FIG. 9 shows the effect of the Aspergillus fumigatus
cellobiohydrolase I variants AC1-621, AC1-959, AC1-964, AC1-971,
and AC1-974, and the Aspergillus fumigatus cellobiohydrolase I
AC1-596 wild-type parent on hydrolysis of milled unwashed
pretreated corn stover (PCS) by a cellulolytic enzyme composition
at 50.degree. C., 55.degree. C. and 60.degree. C.
DEFINITIONS
[0024] Acetylxylan esterase: The term "acetylxylan esterase" means
a carboxylesterase (EC 3.1.1.72) that catalyzes the hydrolysis of
acetyl groups from polymeric xylan, acetylated xylose, acetylated
glucose, alpha-napthyl acetate, and p-nitrophenyl acetate. For
purposes of the present invention, acetylxylan esterase activity is
determined using 0.5 mM p-nitrophenylacetate as substrate in 50 mM
sodium acetate pH 5.0 containing 0.01% TWEEN.TM. 20
(polyoxyethylene sorbitan monolaurate). One unit of acetylxylan
esterase is defined as the amount of enzyme capable of releasing 1
.mu.mole of p-nitrophenolate anion per minute at pH 5, 25.degree.
C.
[0025] Allelic variant: The term "allelic variant" means any of two
or more alternative forms of a gene occupying the same chromosomal
locus. Allelic variation arises naturally through mutation, and may
result in polymorphism within populations. Gene mutations can be
silent (no change in the encoded polypeptide) or may encode
polypeptides having altered amino acid sequences. An allelic
variant of a polypeptide is a polypeptide encoded by an allelic
variant of a gene.
[0026] Alpha-L-arabinofuranosidase: The term
"alpha-L-arabinofuranosidase" means an alpha-L-arabinofuranoside
arabinofuranohydrolase (EC 3.2.1.55) that catalyzes the hydrolysis
of terminal non-reducing alpha-L-arabinofuranoside residues in
alpha-L-arabinosides. The enzyme acts on
alpha-L-arabinofuranosides, alpha-L-arabinans containing (1,3)-
and/or (1,5)-linkages, arabinoxylans, and arabinogalactans.
Alpha-L-arabinofuranosidase is also known as arabinosidase,
alpha-arabinosidase, alpha-L-arabinosidase,
alpha-arabinofuranosidase, polysaccharide
alpha-L-arabinofuranosidase, alpha-L-arabinofuranoside hydrolase,
L-arabinosidase, or alpha-L-arabinanase. For purposes of the
present invention, alpha-L-arabinofuranosidase activity is
determined using 5 mg of medium viscosity wheat arabinoxylan
(Megazyme International Ireland, Ltd., Bray, Co. Wicklow, Ireland)
per ml of 100 mM sodium acetate pH 5 in a total volume of 200 .mu.l
for 30 minutes at 40.degree. C. followed by arabinose analysis by
AMINEX.RTM. HPX-87H column chromatography (Bio-Rad Laboratories,
Inc., Hercules, Calif., USA).
[0027] Alpha-glucuronidase: The term "alpha-glucuronidase" means an
alpha-D-glucosiduronate glucuronohydrolase (EC 3.2.1.139) that
catalyzes the hydrolysis of an alpha-D-glucuronoside to
D-glucuronate and an alcohol. For purposes of the present
invention, alpha-glucuronidase activity is determined according to
de Vries, 1998, J. Bacteriol. 180: 243-249. One unit of
alpha-glucuronidase equals the amount of enzyme capable of
releasing 1 .mu.mole of glucuronic or 4-O-methylglucuronic acid per
minute at pH 5, 40.degree. C.
[0028] Beta-glucosidase: The term "beta-glucosidase" means a
beta-D-glucoside glucohydrolase (E.C. 3.2.1.21) that catalyzes the
hydrolysis of terminal non-reducing beta-D-glucose residues with
the release of beta-D-glucose. For purposes of the present
invention, beta-glucosidase activity is determined using
p-nitrophenyl-beta-D-glucopyranoside as substrate according to the
procedure of Venturi et al., 2002, J. Basic Microbiol. 42: 55-66.
One unit of beta-glucosidase is defined as 1.0 .mu.mole of
p-nitrophenolate anion produced per minute at 25.degree. C., pH 4.8
from 1 mM p-nitrophenyl-beta-D-glucopyranoside as substrate in 50
mM sodium citrate containing 0.01% TWEEN.RTM. 20.
[0029] Beta-xylosidase: The term "beta-xylosidase" means a
beta-D-xyloside xylohydrolase (E.C. 3.2.1.37) that catalyzes the
exo-hydrolysis of short beta (1.fwdarw.4)-xylooligosaccharides to
remove successive D-xylose residues from non-reducing termini. For
purposes of the present invention, beta-xylosidase activity is
determined using 1 mM p-nitrophenyl-beta-D-xyloside as substrate in
100 mM sodium citrate containing 0.01% TWEEN.RTM. 20 at pH 5,
40.degree. C. One unit of beta-xylosidase is defined as 1.0
.mu.mole of p-nitrophenolate anion produced per minute at
40.degree. C., pH 5 from 1 mM p-nitrophenyl-beta-D-xyloside as
substrate in 100 mM sodium citrate containing 0.01% TWEEN.RTM.
20.
[0030] Carbohydrate binding module: The term "carbohydrate binding
module" means a domain within a carbohydrate-active enzyme that
provides carbohydrate-binding activity (Boraston et al., 2004,
Biochem. J. 383: 769-781). A majority of known carbohydrate binding
modules (CBMs) are contiguous amino acid sequences with a discrete
fold. The carbohydrate binding module (CBM) is typically found
either at the N-terminal or at the C-terminal extremity of an
enzyme. The term "carbohydrate binding module" is also used
interchangedly herein with the term "carbohydrate binding
domain".
[0031] Catalytic domain: The term "catalytic domain" means the
region of an enzyme containing the catalytic machinery of the
enzyme. In one aspect, the catalytic domain is amino acids 1 to 437
of SEQ ID NO: 4. In another aspect, the catalytic domain is amino
acids 1 to 429 of SEQ ID NO: 6. In another aspect, the catalytic
domain is amino acids 1 to 440 of SEQ ID NO: 8. In another aspect,
the catalytic domain is amino acids 1 to 437 of SEQ ID NO: 10. In
another aspect, the catalytic domain is amino acids 1 to 437 of SEQ
ID NO: 12. In another aspect, the catalytic domain is amino acids 1
to 438 of SEQ ID NO: 14. In another aspect, the catalytic domain is
amino acids 1 to 437 of SEQ ID NO: 16. In another aspect, the
catalytic domain is amino acids 1 to 430 of SEQ ID NO: 18. In
another aspect, the catalytic domain is amino acids 1 to 433 of SEQ
ID NO: 20. In another aspect, the catalytic domain is amino acids 1
to 438 of SEQ ID NO: 22. In another aspect, the catalytic domain is
amino acids 1 to 437 of SEQ ID NO: 26. In another aspect, the
catalytic domain is amino acids 1 to 437 of SEQ ID NO: 28. In
another aspect, the catalytic domain is amino acids 1 to 437 of SEQ
ID NO: 38. In another aspect, the catalytic domain is amino acids 1
to 437 of SEQ ID NO: 42. In another aspect, the catalytic domain is
amino acids 1 to 437 of SEQ ID NO: 46. In another aspect, the
catalytic domain is amino acids 1 to 437 of SEQ ID NO: 48. In
another aspect, the catalytic domain is amino acids 1 to 437 of SEQ
ID NO: 52. In another aspect, the catalytic domain is amino acids 1
to 437 of SEQ ID NO: 57. In another aspect, the catalytic domain is
amino acids 1 to 437 of SEQ ID NO: 63. In another aspect, the
catalytic domain is amino acids 1 to 437 of SEQ ID NO: 67. In
another aspect, the catalytic domain is amino acids 1 to 437 of SEQ
ID NO: 69. In another aspect, the catalytic domain is amino acids 1
to 437 of SEQ ID NO: 71. In another aspect, the catalytic domain is
amino acids 1 to 437 of SEQ ID NO: 73. In another aspect, the
catalytic domain is amino acids 1 to 437 of SEQ ID NO: 75. In
another aspect, the catalytic domain is amino acids 1 to 437 of SEQ
ID NO: 77. In another aspect, the catalytic domain is amino acids 1
to 437 of SEQ ID NO: 79. In another aspect, the catalytic domain is
amino acids 1 to 437 of SEQ ID NO: 81. In another aspect, the
catalytic domain is amino acids 1 to 437 of SEQ ID NO: 83. In
another aspect, the catalytic domain is amino acids 1 to 437 of SEQ
ID NO: 85.
[0032] Catalytic domain coding sequence: The term "catalytic domain
coding sequence" means a polynucleotide that encodes a domain
catalyzing cellobiohydrolase activity. In one aspect, the catalytic
domain coding sequence is nucleotides 79 to 1389 of SEQ ID NO: 1.
In one aspect, the catalytic domain coding sequence is nucleotides
79 to 1389 of SEQ ID NO: 2. In one aspect, the catalytic domain
coding sequence is nucleotides 79 to 1389 of SEQ ID NO: 3. In
another aspect, the catalytic domain coding sequence is nucleotides
52 to 1469 of SEQ ID NO: 5. In another aspect, the catalytic domain
coding sequence is nucleotides 52 to 1371 of SEQ ID NO: 7. In
another aspect, the catalytic domain coding sequence is nucleotides
55 to 1425 of SEQ ID NO: 9. In another aspect, the catalytic domain
coding sequence is nucleotides 76 to 1386 of SEQ ID NO: 11. In
another aspect, the catalytic domain coding sequence is nucleotides
76 to 1389 of SEQ ID NO: 13. In another aspect, the catalytic
domain coding sequence is nucleotides 55 to 1504 of SEQ ID NO: 15.
In another aspect, the catalytic domain coding sequence is
nucleotides 61 to 1350 of SEQ ID NO: 17. In another aspect, the
catalytic domain coding sequence is nucleotides 55 to 1353 of SEQ
ID NO: 19. In another aspect, the catalytic domain coding sequence
is nucleotides 55 to 1368 of SEQ ID NO: 21. In one aspect, the
catalytic domain coding sequence is nucleotides 79 to 1389 of SEQ
ID NO: 25. In one aspect, the catalytic domain coding sequence is
nucleotides 79 to 1389 of SEQ ID NO: 27. In one aspect, the
catalytic domain coding sequence is nucleotides 79 to 1389 of SEQ
ID NO: 37. In one aspect, the catalytic domain coding sequence is
nucleotides 79 to 1389 of SEQ ID NO: 41. In one aspect, the
catalytic domain coding sequence is nucleotides 79 to 1389 of SEQ
ID NO: 45. In one aspect, the catalytic domain coding sequence is
nucleotides 79 to 1389 of SEQ ID NO: 47.
[0033] cDNA: The term "cDNA" means a DNA molecule that can be
prepared by reverse transcription from a mature, spliced, mRNA
molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks
intron sequences that may be present in the corresponding genomic
DNA. The initial, primary RNA transcript is a precursor to mRNA
that is processed through a series of steps, including splicing,
before appearing as mature spliced mRNA.
[0034] Cellobiohydrolase: The term "cellobiohydrolase" means a
1,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91 and E.C.
3.2.1.176) that catalyzes the hydrolysis of 1,4-beta-D-glucosidic
linkages in cellulose, cellooligosaccharides, or any
beta-1,4-linked glucose containing polymer, releasing cellobiose
from the reducing end (cellobiohydrolase I) or non-reducing end
(cellobiohydrolase II) of the chain (Teeri, 1997, Trends in
Biotechnology 15: 160-167; Teeri et al., 1998, Biochem. Soc. Trans.
26: 173-178). Cellobiohydrolase activity is determined according to
the procedures described by Lever et al., 1972, Anal. Biochem. 47:
273-279; van Tilbeurgh et al., 1982, FEBS Letters, 149: 152-156;
van Tilbeurgh and Claeyssens, 1985, FEBS Letters, 187: 283-288; and
Tomme et al., 1988, Eur. J. Biochem. 170: 575-581. In the present
invention, cellobiohydrolase activity is preferably determined
according to Examples 8 and 9 herein.
[0035] Cellulolytic enzyme or cellulase: The term "cellulolytic
enzyme" or "cellulase" means one or more (e.g., several) enzymes
that hydrolyze a cellulosic material. Such enzymes include
endoglucanase(s), cellobiohydrolase(s), beta-glucosidase(s), or
combinations thereof. The two basic approaches for measuring
cellulolytic enzyme activity include: (1) measuring the total
cellulolytic enzyme activity, and (2) measuring the individual
cellulolytic enzyme activities (endoglucanases, cellobiohydrolases,
and beta-glucosidases) as reviewed in Zhang et al., 2006,
Biotechnology Advances 24: 452-481. Total cellulolytic activity is
usually measured using insoluble substrates, including Whatman No1
filter paper, microcrystalline cellulose, bacterial cellulose,
algal cellulose, cotton, pretreated lignocellulose, etc. The most
common total cellulolytic activity assay is the filter paper assay
using Whatman No1 filter paper as the substrate. The assay was
established by the International Union of Pure and Applied
Chemistry (IUPAC) (Ghose, 1987, Pure Appl. Chem. 59: 257-68).
[0036] For purposes of the present invention, cellulolytic enzyme
activity is determined by measuring the increase in hydrolysis of a
cellulosic material by cellulolytic enzyme(s) under the following
conditions: 1-50 mg of cellulolytic enzyme protein/g of cellulose
in PCS (or other pretreated cellulosic material) for 3-7 days at a
suitable temperature such as 25.degree. C.-80.degree. C., e.g.,
30.degree. C., 35.degree. C., 40.degree. C., 45.degree. C.,
50.degree. C., 55.degree. C., 60.degree. C., 65.degree. C., or
70.degree. C., and a suitable pH such as 4-9, e.g., 5.0, 5.5, 6.0,
6.5, or 7.0, compared to a control hydrolysis without addition of
cellulolytic enzyme protein. Typical conditions are 1 ml reactions,
washed or unwashed PCS, 5% insoluble solids (dry weight), 50 mM
sodium acetate pH 5, 1 mM MnSO.sub.4, 50.degree. C., 55.degree. C.,
or 60.degree. C., 72 hours, sugar analysis by AMINEX.RTM. HPX-87H
column chromatography (Bio-Rad Laboratories, Inc., Hercules,
Calif., USA).
[0037] Cellulosic material: The term "cellulosic material" means
any material containing cellulose. The predominant polysaccharide
in the primary cell wall of biomass is cellulose, the second most
abundant is hemicellulose, and the third is pectin. The secondary
cell wall, produced after the cell has stopped growing, also
contains polysaccharides and is strengthened by polymeric lignin
covalently cross-linked to hemicellulose. Cellulose is a
homopolymer of anhydrocellobiose and thus a linear
beta-(1-4)-D-glucan, while hemicelluloses include a variety of
compounds, such as xylans, xyloglucans, arabinoxylans, and mannans
in complex branched structures with a spectrum of substituents.
Although generally polymorphous, cellulose is found in plant tissue
primarily as an insoluble crystalline matrix of parallel glucan
chains. Hemicelluloses usually hydrogen bond to cellulose, as well
as to other hemicelluloses, which help stabilize the cell wall
matrix.
[0038] Cellulose is generally found, for example, in the stems,
leaves, hulls, husks, and cobs of plants or leaves, branches, and
wood of trees. The cellulosic material can be, but is not limited
to, agricultural residue, herbaceous material (including energy
crops), municipal solid waste, pulp and paper mill residue, waste
paper, and wood (including forestry residue) (see, for example,
Wiselogel et al., 1995, in Handbook on Bioethanol (Charles E.
Wyman, editor), pp. 105-118, Taylor & Francis, Washington D.C.;
Wyman, 1994, Bioresource Technology 50: 3-16; Lynd, 1990, Applied
Biochemistry and Biotechnology 24/25: 695-719; Mosier et al., 1999,
Recent Progress in Bioconversion of Lignocellulosics, in Advances
in Biochemical Engineering/Biotechnology, T. Scheper, managing
editor, Volume 65, pp. 23-40, Springer-Verlag, New York). It is
understood herein that the cellulose may be in the form of
lignocellulose, a plant cell wall material containing lignin,
cellulose, and hemicellulose in a mixed matrix. In one aspect, the
cellulosic material is any biomass material. In another aspect, the
cellulosic material is lignocellulose, which comprises cellulose,
hemicelluloses, and lignin.
[0039] In an embodiment, the cellulosic material is agricultural
residue, herbaceous material (including energy crops), municipal
solid waste, pulp and paper mill residue, waste paper, or wood
(including forestry residue).
[0040] In another embodiment, the cellulosic material is arundo,
bagasse, bamboo, corn cob, corn fiber, corn stover, miscanthus,
rice straw, switchgrass, or wheat straw.
[0041] In another embodiment, the cellulosic material is aspen,
eucalyptus, fir, pine, poplar, spruce, or willow.
[0042] In another embodiment, the cellulosic material is algal
cellulose, bacterial cellulose, cotton linter, filter paper,
microcrystalline cellulose (e.g., AVICEL.RTM.), or phosphoric-acid
treated cellulose.
[0043] In another embodiment, the cellulosic material is an aquatic
biomass. As used herein the term "aquatic biomass" means biomass
produced in an aquatic environment by a photosynthesis process. The
aquatic biomass can be algae, emergent plants, floating-leaf
plants, or submerged plants.
[0044] The cellulosic material may be used as is or may be
subjected to pretreatment, using conventional methods known in the
art, as described herein. In a preferred aspect, the cellulosic
material is pretreated.
[0045] Coding sequence: The term "coding sequence" means a
polynucleotide, which directly specifies the amino acid sequence of
a variant. The boundaries of the coding sequence are generally
determined by an open reading frame, which begins with a start
codon such as ATG, GTG or TTG and ends with a stop codon such as
TAA, TAG, or TGA. The coding sequence may be a genomic DNA, cDNA,
synthetic DNA, or a combination thereof.
[0046] Control sequences: The term "control sequences" means
nucleic acid sequences necessary for expression of a polynucleotide
encoding a variant of the present invention. Each control sequence
may be native (i.e., from the same gene) or foreign (i.e., from a
different gene) to the polynucleotide encoding the variant or
native or foreign to each other. Such control sequences include,
but are not limited to, a leader, polyadenylation sequence,
propeptide sequence, promoter, signal peptide sequence, and
transcription terminator. At a minimum, the control sequences
include a promoter, and transcriptional and translational stop
signals. The control sequences may be provided with linkers for the
purpose of introducing specific restriction sites facilitating
ligation of the control sequences with the coding region of the
polynucleotide encoding a variant.
[0047] Endoglucanase: The term "endoglucanase" means a
4-(1,3;1,4)-beta-D-glucan 4-glucanohydrolase (E.C. 3.2.1.4) that
catalyzes endohydrolysis of 1,4-beta-D-glycosidic linkages in
cellulose, cellulose derivatives (such as carboxymethyl cellulose
and hydroxyethyl cellulose), lichenin, beta-1,4 bonds in mixed
beta-1,3-1,4 glucans such as cereal beta-D-glucans or xyloglucans,
and other plant material containing cellulosic components.
Endoglucanase activity can be determined by measuring reduction in
substrate viscosity or increase in reducing ends determined by a
reducing sugar assay (Zhang et al., 2006, Biotechnology Advances
24: 452-481). For purposes of the present invention, endoglucanase
activity is determined using carboxymethyl cellulose (CMC) as
substrate according to the procedure of Ghose, 1987, Pure and Appl.
Chem. 59: 257-268, at pH 5, 40.degree. C.
[0048] Expression: The term "expression" includes any step involved
in the production of a variant including, but not limited to,
transcription, post-transcriptional modification, translation,
post-translational modification, and secretion.
[0049] Expression vector: The term "expression vector" means a
linear or circular DNA molecule that comprises a polynucleotide
encoding a variant and is operably linked to control sequences that
provide for its expression.
[0050] Family 61 glycoside hydrolase: The term "Family 61 glycoside
hydrolase" or "Family GH61" or "GH61" means a polypeptide falling
into the glycoside hydrolase Family 61 according to Henrissat,
1991, Biochem. J. 280: 309-316, and Henrissat, and Bairoch, 1996,
Biochem. J. 316: 695-696. The GH61 polypeptides have recently been
classified as lytic polysaccharide monooxygenases (Quinlan et al.,
2011, Proc. Natl. Acad. Sci. USA 208: 15079-15084; Phillips et al.,
2011, ACS Chem. Biol. 6: 1399-1406; Lin et al., 2012, Structure 20:
1051-1061) and are designated "Auxiliary Activity 9" or "AA9"
polypeptides.
[0051] Feruloyl esterase: The term "feruloyl esterase" means a
4-hydroxy-3-methoxycinnamoyl-sugar hydrolase (EC 3.1.1.73) that
catalyzes the hydrolysis of 4-hydroxy-3-methoxycinnamoyl (feruloyl)
groups from esterified sugar, which is usually arabinose in natural
biomass substrates, to produce ferulate
(4-hydroxy-3-methoxycinnamate). Feruloyl esterase (FAE) is also
known as ferulic acid esterase, hydroxycinnamoyl esterase, FAE-III,
cinnamoyl ester hydrolase, FAEA, cinnAE, FAE-I, or FAE-II. For
purposes of the present invention, feruloyl esterase activity is
determined using 0.5 mM p-nitrophenylferulate as substrate in 50 mM
sodium acetate pH 5.0. One unit of feruloyl esterase equals the
amount of enzyme capable of releasing 1 .mu.mole of
p-nitrophenolate anion per minute at pH 5, 25.degree. C.
[0052] Fragment: The term "fragment" means a polypeptide having one
or more (e.g., several) amino acids absent from the amino and/or
carboxyl terminus of the referenced polypeptide sequence; wherein
the fragment has cellobiohydrolase activity. In one aspect, the
number of amino acid residues in the fragment is at least 75%,
e.g., at least 80%, 85%, 90%, or 95% of the number of amino acid
residues of the referenced polypeptide sequence or a variant
thereof (e.g., SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,
26, 28, 38, 42, 46, 48, 52, 57, 63, 67, 69, 71, 73, 75, 77, 79, 81,
83, 85, or a variant thereof).
[0053] Hemicellulolytic enzyme or hemicellulase: The term
"hemicellulolytic enzyme" or "hemicellulase" means one or more
(e.g., several) enzymes that hydrolyze a hemicellulosic material.
See, for example, Shallom and Shoham, 2003, Current Opinion In
Microbiology 6(3): 219-228). Hemicellulases are key components in
the degradation of plant biomass. Examples of hemicellulases
include, but are not limited to, an acetylmannan esterase, an
acetylxylan esterase, an arabinanase, an arabinofuranosidase, a
coumaric acid esterase, a feruloyl esterase, a galactosidase, a
glucuronidase, a glucuronoyl esterase, a mannanase, a mannosidase,
a xylanase, and a xylosidase. The substrates for these enzymes,
hemicelluloses, are a heterogeneous group of branched and linear
polysaccharides that are bound via hydrogen bonds to the cellulose
microfibrils in the plant cell wall, crosslinking them into a
robust network. Hemicelluloses are also covalently attached to
lignin, forming together with cellulose a highly complex structure.
The variable structure and organization of hemicelluloses require
the concerted action of many enzymes for its complete degradation.
The catalytic modules of hemicellulases are either glycoside
hydrolases (GHs) that hydrolyze glycosidic bonds, or carbohydrate
esterases (CEs), which hydrolyze ester linkages of acetate or
ferulic acid side groups. These catalytic modules, based on
homology of their primary sequence, can be assigned into GH and CE
families. Some families, with an overall similar fold, can be
further grouped into clans, marked alphabetically (e.g., GH-A). A
most informative and updated classification of these and other
carbohydrate active enzymes is available in the Carbohydrate-Active
Enzymes (CAZy) database. Hemicellulolytic enzyme activities can be
measured according to Ghose and Bisaria, 1987, Pure & Appl.
Chem. 59: 1739-1752, at a suitable temperature such as 40.degree.
C.-80.degree. C., e.g., 50.degree. C., 55.degree. C., 60.degree.
C., 65.degree. C., or 70.degree. C., and a suitable pH such as 4-9,
e.g., 5.0, 5.5, 6.0, 6.5, or 7.0.
[0054] High stringency conditions: The term "high stringency
conditions" means for probes of at least 100 nucleotides in length,
prehybridization and hybridization at 42.degree. C. in
5.times.SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured
salmon sperm DNA, and 50% formamide, following standard Southern
blotting procedures for 12 to 24 hours. The carrier material is
finally washed three times each for 15 minutes using 0.2.times.SSC,
0.2% SDS at 65.degree. C.
[0055] Host cell: The term "host cell" means any cell type that is
susceptible to transformation, transfection, transduction, or the
like with a nucleic acid construct or expression vector comprising
a polynucleotide of the present invention. The term "host cell"
encompasses any progeny of a parent cell that is not identical to
the parent cell due to mutations that occur during replication.
[0056] Improved property: The term "improved property" means a
characteristic associated with a variant that is improved compared
to the parent. Such an improved property is preferably increased
specific performance.
[0057] Increased specific performance: The term "increased specific
performance" by a variant of the present invention means improved
conversion of a cellulosic material to a product, as compared to
the same level of conversion by the parent. Increased specific
performance is determined per unit protein (e.g., mg protein, or
.mu.mole protein). The increased specific performance of the
variant relative to the parent can be assessed, for example, under
one or more (e.g., several) conditions of pH, temperature, and
substrate concentration. In one aspect, the product is glucose. In
another aspect, the product is cellobiose. In another aspect, the
product is glucose+cellobiose.
[0058] In one aspect, the condition is pH. For example, the pH can
be any pH in the range of 3 to 7, e.g., 3.0, 3.5, 4.0, 4.5, 5.0,
5.5, 6.0, 6.5, or 7.0 (or in between). Any suitable buffer for
achieving the desired pH can be used.
[0059] In another aspect, the condition is temperature. For
example, the temperature can be any temperature in the range of
25.degree. C. to 90.degree. C., e.g., 25, 30, 35, 40, 45, 50, 55,
60, 65, 70, 75, 80, 85, or 90.degree. C. (or in between).
[0060] In another aspect, the condition is substrate concentration.
Any cellulosic material defined herein can be used as the
substrate. In one aspect, the substrate concentration is measured
as the dry solids content. The dry solids content is preferably in
the range of about 1 to about 50 wt %, e.g., about 5 to about 45 wt
%, about 10 to about 40 wt %, or about 20 to about 30 wt %. In
another aspect, the substrate concentration is measured as the
insoluble glucan content. The insoluble glucan content is
preferably in the range of about 2.5 to about 25 wt %, e.g., about
5 to about 20 wt % or about 10 to about 15 wt %.
[0061] In another aspect, a combination of two or more (e.g.,
several) of the above conditions are used to determine the
increased specific performance of the variant relative to the
parent, such as any temperature in the range of 25.degree. C. to
90.degree. C., e.g., 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,
80, 85, or 90.degree. C. (or in between) at a pH in the range of 3
to 7, e.g., 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, or 7.0 (or in
between).
[0062] The increased specific performance of the variant relative
to the parent can be determined using any enzyme assay known in the
art for cellobiohydrolases as described herein. Alternatively, the
increased specific performance of the variant relative to the
parent can be determined using the assays described in Examples 8
and 9.
[0063] In another aspect, the specific performance of the variant
is at least 1.01-fold, e.g., at least 1.02-fold, at least
1.03-fold, at least 1.04-fold, at least 1.05-fold, at least
1.06-fold, at least 1.07-fold, at least 1.08-fold, at least
1.09-fold, at least 1.1-fold, at least 1.2-fold, at least 1.3-fold,
at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least
1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2-fold, at
least 2.1-fold, at least 2.2-fold, at least 2.3-fold, at least
2.4-fold, at least 2.5-fold, at least 5-fold, at least 10-fold, at
least 15-fold, at least 20-fold, at least 25-fold, and at least
50-fold higher than the specific performance of the parent.
[0064] Isolated: The term "isolated" means a substance in a form or
environment that does not occur in nature. Non-limiting examples of
isolated substances include (1) any non-naturally occurring
substance, (2) any substance including, but not limited to, any
enzyme, variant, nucleic acid, protein, peptide or cofactor, that
is at least partially removed from one or more or all of the
naturally occurring constituents with which it is associated in
nature; (3) any substance modified by the hand of man relative to
that substance found in nature; or (4) any substance modified by
increasing the amount of the substance relative to other components
with which it is naturally associated (e.g., recombinant production
in a host cell; multiple copies of a gene encoding the substance;
and use of a stronger promoter than the promoter naturally
associated with the gene encoding the substance).
[0065] Low stringency conditions: The term "low stringency
conditions" means for probes of at least 100 nucleotides in length,
prehybridization and hybridization at 42.degree. C. in
5.times.SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured
salmon sperm DNA, and 25% formamide, following standard Southern
blotting procedures for 12 to 24 hours. The carrier material is
finally washed three times each for 15 minutes using 0.2.times.SSC,
0.2% SDS at 50.degree. C.
[0066] Mature polypeptide: The term "mature polypeptide" means a
polypeptide in its final form following translation and any
post-translational modifications, such as N-terminal processing,
C-terminal truncation, glycosylation, phosphorylation, etc. In one
aspect, the mature polypeptide is amino acids 1 to 506 of SEQ ID
NO: 4 based on the SignalP 3.0 program (Bendtsen et al., 2004, J.
Mol. Biol. 340: 783-795) that predicts amino acids -1 to -26 of SEQ
ID NO: 4 are a signal peptide. In another aspect, the mature
polypeptide is amino acids 1 to 497 of SEQ ID NO: 6 based on the
SignalP 3.0 program that predicts amino acids -1 to -17 of SEQ ID
NO: 6 are a signal peptide. In another aspect, the mature
polypeptide is amino acids 1 to 440 of SEQ ID NO: 8 based on the
SignalP 3.0 program that predicts amino acids -1 to -17 of SEQ ID
NO: 8 are a signal peptide. In another aspect, the mature
polypeptide is amino acids 1 to 437 of SEQ ID NO: 10 based on the
SignalP 3.0 program that predicts amino acids -1 to -18 of SEQ ID
NO: 10 are a signal peptide. In another aspect, the mature
polypeptide is amino acids 1 to 507 of SEQ ID NO: 12 based on the
SignalP 3.0 program that predicts amino acids -1 to -25 of SEQ ID
NO: 12 are a signal peptide. In another aspect, the mature
polypeptide is amino acids 1 to 507 of SEQ ID NO: 14 based on the
SignalP 3.0 program that predicts amino acids -1 to -25 of SEQ ID
NO: 14 are a signal peptide. In another aspect, the mature
polypeptide is amino acids 1 to 437 of SEQ ID NO: 16 based on the
SignalP 3.0 program that predicts amino acids -1 to -18 of SEQ ID
NO: 16 are a signal peptide. In another aspect, the mature
polypeptide is amino acids 1 to 430 of SEQ ID NO: 18 based on the
SignalP 3.0 program that predicts amino acids -1 to -20 of SEQ ID
NO: 18 are a signal peptide. In another aspect, the mature
polypeptide is amino acids 1 to 511 of SEQ ID NO: 20 based on the
SignalP 3.0 program that predicts amino acids -1 to -18 of SEQ ID
NO: 20 are a signal peptide. In another aspect, the mature
polypeptide is amino acids 1 to 507 of SEQ ID NO: 22 based on the
SignalP 3.0 program that predicts amino acids -1 to -18 of SEQ ID
NO: 22 are a signal peptide. In another aspect, the mature
polypeptide is amino acids 1 to 501 of SEQ ID NO: 24 based on the
SignalP 3.0 program that predicts amino acids -1 to -18 of SEQ ID
NO: 24 are a signal peptide. In another aspect, the mature
polypeptide is amino acids 1 to 506 of SEQ ID NO: 26 based on the
SignalP 3.0 program that predicts amino acids -1 to -26 of SEQ ID
NO: 26 are a signal peptide. In another aspect, the mature
polypeptide is amino acids 1 to 506 of SEQ ID NO: 28 based on the
SignalP 3.0 program that predicts amino acids -1 to -26 of SEQ ID
NO: 28 are a signal peptide. In another aspect, the mature
polypeptide is amino acids 1 to 506 of SEQ ID NO: 38 based on the
SignalP 3.0 program that predicts amino acids -1 to -26 of SEQ ID
NO: 38 are a signal peptide. In another aspect, the mature
polypeptide is amino acids 1 to 506 of SEQ ID NO: 42 based on the
SignalP 3.0 program that predicts amino acids -1 to -26 of SEQ ID
NO: 42 are a signal peptide. In another aspect, the mature
polypeptide is amino acids 1 to 506 of SEQ ID NO: 46 based on the
SignalP 3.0 program that predicts amino acids -1 to -26 of SEQ ID
NO: 46 are a signal peptide. In another aspect, the mature
polypeptide is amino acids 1 to 506 of SEQ ID NO: 48 based on the
SignalP 3.0 program that predicts amino acids -1 to -26 of SEQ ID
NO: 48 are a signal peptide. In another aspect, the mature
polypeptide is amino acids 1 to 503 of SEQ ID NO: 57 based on the
SignalP 3.0 program that predicts amino acids -1 to -18 of SEQ ID
NO: 57 are a signal peptide. In another aspect, the mature
polypeptide is amino acids 1 to 503 of SEQ ID NO: 63 based on the
SignalP 3.0 program that predicts amino acids -1 to -18 of SEQ ID
NO: 63 are a signal peptide. In another aspect, the mature
polypeptide is amino acids 1 to 506 of SEQ ID NO: 67 based on the
SignalP 3.0 program that predicts amino acids -1 to -26 of SEQ ID
NO: 67 are a signal peptide. In another aspect, the mature
polypeptide is amino acids 1 to 506 of SEQ ID NO: 69 based on the
SignalP 3.0 program that predicts amino acids -1 to -26 of SEQ ID
NO: 69 are a signal peptide. In another aspect, the mature
polypeptide is amino acids 1 to 506 of SEQ ID NO: 71 based on the
SignalP 3.0 program that predicts amino acids -1 to -26 of SEQ ID
NO: 71 are a signal peptide. In another aspect, the mature
polypeptide is amino acids 1 to 506 of SEQ ID NO: 73 based on the
SignalP 3.0 program that predicts amino acids -1 to -26 of SEQ ID
NO: 73 are a signal peptide. In another aspect, the mature
polypeptide is amino acids 1 to 506 of SEQ ID NO: 75 based on the
SignalP 3.0 program that predicts amino acids -1 to -26 of SEQ ID
NO: 75 are a signal peptide. In another aspect, the mature
polypeptide is amino acids 1 to 506 of SEQ ID NO: 77 based on the
SignalP 3.0 program that predicts amino acids -1 to -26 of SEQ ID
NO: 77 are a signal peptide. In another aspect, the mature
polypeptide is amino acids 1 to 506 of SEQ ID NO: 79 based on the
SignalP 3.0 program that predicts amino acids -1 to -26 of SEQ ID
NO: 79 are a signal peptide. In another aspect, the mature
polypeptide is amino acids 1 to 506 of SEQ ID NO: 81 based on the
SignalP 3.0 program that predicts amino acids -1 to -26 of SEQ ID
NO: 81 are a signal peptide. In another aspect, the mature
polypeptide is amino acids 1 to 506 of SEQ ID NO: 83 based on the
SignalP 3.0 program that predicts amino acids -1 to -26 of SEQ ID
NO: 83 are a signal peptide. In another aspect, the mature
polypeptide is amino acids 1 to 506 of SEQ ID NO: 85 based on the
SignalP 3.0 program that predicts amino acids -1 to -26 of SEQ ID
NO: 85 are a signal peptide. It is known in the art that a host
cell may produce a mixture of two of more different mature
polypeptides (i.e., with a different C-terminal and/or N-terminal
amino acid) expressed by the same polynucleotide. It is also known
in the art that different host cells may process polypeptides
differently, and thus, one host cell expressing a polynucleotide
may produce a different mature polypeptide (e.g., having a
different C-terminal and/or N-terminal amino acid) as compared to
another host cell expressing the same polynucleotide.
[0067] Mature polypeptide coding sequence: The term "mature
polypeptide coding sequence" means a polynucleotide that encodes a
mature polypeptide having cellobiohydrolase activity. In one
aspect, the mature polypeptide coding sequence is nucleotides 79 to
1596 of SEQ ID NO: 1 (without the stop codon) based on SignalP 3.0
program (Bendtsen et al., 2004, supra) that predicts nucleotides 1
to 78 of SEQ ID NO: 1 encode a signal peptide. In another aspect,
the mature polypeptide coding sequence is nucleotides 79 to 1596 of
SEQ ID NO: 2 (without the stop codon) based on SignalP 3.0 program
that predicts nucleotides 1 to 78 of SEQ ID NO: 2 encode a signal
peptide. In another aspect, the mature polypeptide coding sequence
is nucleotides 79 to 1596 of SEQ ID NO: 3 (without the stop codon)
based on SignalP 3.0 program that predicts nucleotides 1 to 78 of
SEQ ID NO: 3 encode a signal peptide. In another aspect, the mature
polypeptide coding sequence is nucleotides 52 to 1673 of SEQ ID NO:
5 (without the stop codon) based on SignalP 3.0 program that
predicts nucleotides 1 to 51 of SEQ ID NO: 5 encode a signal
peptide. In another aspect, the mature polypeptide coding sequence
is nucleotides 52 to 1371 of SEQ ID NO: 7 (without the stop codon)
based on SignalP 3.0 program that predicts nucleotides 1 to 51 of
SEQ ID NO: 7 encode a signal peptide. In another aspect, the mature
polypeptide coding sequence is nucleotides 55 to 1425 of SEQ ID NO:
9 (without the stop codon) based on SignalP 3.0 program that
predicts nucleotides 1 to 54 of SEQ ID NO: 9 encode a signal
peptide. In another aspect, the mature polypeptide coding sequence
is nucleotides 76 to 1596 of SEQ ID NO: 11 (without the stop codon)
based on SignalP 3.0 program that predicts nucleotides 1 to 75 of
SEQ ID NO: 11 encode a signal peptide. In another aspect, the
mature polypeptide coding sequence is nucleotides 76 to 1596 of SEQ
ID NO: 13 (without the stop codon) based on SignalP 3.0 program
that predicts nucleotides 1 to 75 of SEQ ID NO: 13 encode a signal
peptide. In another aspect, the mature polypeptide coding sequence
is nucleotides 55 to 1504 of SEQ ID NO: 15 (without the stop codon)
based on SignalP 3.0 program that predicts nucleotides 1 to 54 of
SEQ ID NO: 15 encode a signal peptide. In another aspect, the
mature polypeptide coding sequence is nucleotides 61 to 1350 of SEQ
ID NO: 17 (without the stop codon) based on SignalP 3.0 program
that predicts nucleotides 1 to 60 of SEQ ID NO: 17 encode a signal
peptide. In another aspect, the mature polypeptide coding sequence
is nucleotides 55 to 1587 of SEQ ID NO: 19 (without the stop codon)
based on SignalP 3.0 program that predicts nucleotides 1 to 54 of
SEQ ID NO: 19 encode a signal peptide. In another aspect, the
mature polypeptide coding sequence is nucleotides 55 to 1575 of SEQ
ID NO: 21 (without the stop codon) based on SignalP 3.0 program
that predicts nucleotides 1 to 54 of SEQ ID NO: 21 encode a signal
peptide. In another aspect, the mature polypeptide coding sequence
is nucleotides 79 to 1596 of SEQ ID NO: 25 (without the stop codon)
based on SignalP 3.0 program that predicts nucleotides 1 to 78 of
SEQ ID NO: 25 encode a signal peptide. In another aspect, the
mature polypeptide coding sequence is nucleotides 79 to 1596 of SEQ
ID NO: 27 (without the stop codon) based on SignalP 3.0 program
that predicts nucleotides 1 to 78 of SEQ ID NO: 27 encode a signal
peptide. In another aspect, the mature polypeptide coding sequence
is nucleotides 79 to 1596 of SEQ ID NO: 37 (without the stop codon)
based on SignalP 3.0 program that predicts nucleotides 1 to 78 of
SEQ ID NO: 37 encode a signal peptide. In another aspect, the
mature polypeptide coding sequence is nucleotides 79 to 1596 of SEQ
ID NO: 41 (without the stop codon) based on SignalP 3.0 program
that predicts nucleotides 1 to 78 of SEQ ID NO: 41 encode a signal
peptide. In another aspect, the mature polypeptide coding sequence
is nucleotides 79 to 1596 of SEQ ID NO: 45 (without the stop codon)
based on SignalP 3.0 program that predicts nucleotides 1 to 78 of
SEQ ID NO: 45 encode a signal peptide. In another aspect, the
mature polypeptide coding sequence is nucleotides 79 to 1596 of SEQ
ID NO: 47 (without the stop codon) based on SignalP 3.0 program
that predicts nucleotides 1 to 78 of SEQ ID NO: 47 encode a signal
peptide. In another aspect, the mature polypeptide coding sequence
is nucleotides 55 to 1563 of SEQ ID NO: 62 (without the stop codon)
based on SignalP 3.0 program that predicts nucleotides 1 to 54 of
SEQ ID NO: 62 encode a signal peptide.
[0068] Medium stringency conditions: The term "medium stringency
conditions" means for probes of at least 100 nucleotides in length,
prehybridization and hybridization at 42.degree. C. in
5.times.SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured
salmon sperm DNA, and 35% formamide, following standard Southern
blotting procedures for 12 to 24 hours. The carrier material is
finally washed three times each for 15 minutes using 0.2.times.SSC,
0.2% SDS at 55.degree. C.
[0069] Medium-high stringency conditions: The term "medium-high
stringency conditions" means for probes of at least 100 nucleotides
in length, prehybridization and hybridization at 42.degree. C. in
5.times.SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured
salmon sperm DNA, and 35% formamide, following standard Southern
blotting procedures for 12 to 24 hours. The carrier material is
finally washed three times each for 15 minutes using 0.2.times.SSC,
0.2% SDS at 60.degree. C.
[0070] Mutant: The term "mutant" means a polynucleotide encoding a
variant.
[0071] Nucleic acid construct: The term "nucleic acid construct"
means a nucleic acid molecule, either single- or double-stranded,
which is isolated from a naturally occurring gene or is modified to
contain segments of nucleic acids in a manner that would not
otherwise exist in nature or which is synthetic, which comprises
one or more control sequences.
[0072] Operably linked: The term "operably linked" means a
configuration in which a control sequence is placed at an
appropriate position relative to the coding sequence of a
polynucleotide such that the control sequence directs expression of
the coding sequence.
[0073] Parent or parent cellobiohydrolase: The term "parent" or
"parent cellobiohydrolase" means a polypeptide having
cellobiohydrolase activity to which an alteration, i.e., a
substitution, insertion, and/or deletion, at one or more (e.g.,
several) positions, is made to produce an enzyme variant of the
present invention. The parent may be a naturally occurring
(wild-type) polypeptide or a variant or fragment thereof.
[0074] Polypeptide having cellulolytic enhancing activity: The term
"polypeptide having cellulolytic enhancing activity" means a GH61
polypeptide or variant thereof that catalyzes the enhancement of
the hydrolysis of a cellulosic material by enzyme having
cellulolytic activity, i.e., a cellulase. For purposes of the
present invention, cellulolytic enhancing activity is determined by
measuring the increase in reducing sugars or the increase of the
total of cellobiose and glucose from the hydrolysis of a cellulosic
material by cellulolytic enzyme under the following conditions:
1-50 mg of total protein/g of cellulose in pretreated corn stover
(PCS), wherein total protein is comprised of 50-99.5% w/w
cellulolytic enzyme protein and 0.5-50% w/w protein of a GH61
polypeptide or variant thereof for 1-7 days at a suitable
temperature, such as 25.degree. C.-80.degree. C., e.g., 30.degree.
C., 35.degree. C., 40.degree. C., 45.degree. C., 50.degree. C.,
55.degree. C., 60.degree. C., 65.degree. C., or 70.degree. C., and
a suitable pH, such as 4-9, e.g., 4.5, 5.0, 5.5, 6.0, 6.5, 7.0,
7.5, 8.0, or 8.5, compared to a control hydrolysis with equal total
protein loading without cellulolytic enhancing activity (1-50 mg of
cellulolytic protein/g of cellulose in PCS) In one aspect, GH61
polypeptide enhancing activity is determined using a mixture of
CELLUCLAST.RTM. 1.5 L (Novozymes A/S, Bagsv.ANG.rd, Denmark) in the
presence of 2-3% of total protein weight Aspergillus oryzae
beta-glucosidase (recombinantly produced in Aspergillus oryzae
according to WO 02/095014) or 2-3% of total protein weight
Aspergillus fumigatus beta-glucosidase (recombinantly produced in
Aspergillus oryzae as described in WO 02/095014) of cellulase
protein loading is used as the source of the cellulolytic
activity.
[0075] Another assay for determining the cellulolytic enhancing
activity of a GH61 polypeptide or variant thereof is to incubate
the GH61 polypeptide or variant with 0.5% phosphoric acid swollen
cellulose (PASC), 100 mM sodium acetate pH 5, 1 mM MnSO.sub.4, 0.1%
gallic acid, 0.025 mg/ml of Aspergillus fumigatus beta-glucosidase,
and 0.01% TRITON.RTM. X100
(4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol) for 24-96
hours at 40.degree. C. followed by determination of the glucose
released from the PASC.
[0076] The GH61 polypeptides or variants thereof having
cellulolytic enhancing activity enhance the hydrolysis of a
cellulosic material catalyzed by enzyme having cellulolytic
activity by reducing the amount of cellulolytic enzyme required to
reach the same degree of hydrolysis preferably at least 1.01-fold,
e.g., at least 1.05-fold, at least 1.10-fold, at least 1.25-fold,
at least 1.5-fold, at least 2-fold, at least 3-fold, at least
4-fold, at least 5-fold, at least 10-fold, or at least 20-fold.
[0077] Pretreated corn stover: The term "Pretreated Corn Stover" or
"PCS" means a cellulosic material derived from corn stover by
treatment with heat and dilute sulfuric acid, alkaline
pretreatment, neutral pretreatment, or any pretreatment known in
the art.
[0078] Sequence identity: The relatedness between two amino acid
sequences or between two nucleotide sequences is described by the
parameter "sequence identity".
[0079] For purposes of the present invention, the sequence identity
between two amino acid sequences is determined using the
Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol.
Biol. 48: 443-453) as implemented in the Needle program of the
EMBOSS package (EMBOSS: The European Molecular Biology Open
Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277),
preferably version 5.0.0 or later. The parameters used are a gap
open penalty of 10, a gap extension penalty of 0.5, and the
EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The
output of Needle labeled "longest identity" (obtained using the
-nobrief option) is used as the percent identity and is calculated
as follows:
(Identical Residues.times.100)/(Length of Alignment-Total Number of
Gaps in Alignment)
[0080] For purposes of the present invention, the sequence identity
between two deoxyribonucleotide sequences is determined using the
Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as
implemented in the Needle program of the EMBOSS package (EMBOSS:
The European Molecular Biology Open Software Suite, Rice et al.,
2000, supra), preferably version 5.0.0 or later. The parameters
used are a gap open penalty of 10, a gap extension penalty of 0.5,
and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution
matrix. The output of Needle labeled "longest identity" (obtained
using the -nobrief option) is used as the percent identity and is
calculated as follows:
(Identical Deoxyribonucleotides.times.100)/(Length of
Alignment-Total Number of Gaps in Alignment)
[0081] Subsequence: The term "subsequence" means a polynucleotide
having one or more (e.g., several) nucleotides absent from the 5'
and/or 3' end of the referenced nucleotide sequence; wherein the
subsequence encodes a fragment having cellobiohydrolase activity.
In one aspect, the number of nucleotides residues in the
subsequence is at least 75%, e.g., at least 80%, 85%, 90%, or 95%
of the number of nucleotide residues in the referenced sequence
(e.g., SEQ ID NO: 1, 2, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 25, 27,
37, 41, 45, 47, 51, 56 or 62).
[0082] Variant: The term "variant" means a polypeptide having
cellobiohydrolase activity comprising an alteration, i.e., a
substitution, insertion, and/or deletion, at one or more (e.g.,
several) positions. A substitution means replacement of the amino
acid occupying a position with a different amino acid; a deletion
means removal of the amino acid occupying a position; and an
insertion means adding an amino acid adjacent to and immediately
following the amino acid occupying a position. The variants of the
present invention have a specific performance which is at least
1.01-fold higher than the specific performance of the parent.
[0083] Very high stringency conditions: The term "very high
stringency conditions" means for probes of at least 100 nucleotides
in length, prehybridization and hybridization at 42.degree. C. in
5.times.SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured
salmon sperm DNA, and 50% formamide, following standard Southern
blotting procedures for 12 to 24 hours. The carrier material is
finally washed three times each for 15 minutes using 0.2.times.SSC,
0.2% SDS at 70.degree. C.
[0084] Very low stringency conditions: The term "very low
stringency conditions" means for probes of at least 100 nucleotides
in length, prehybridization and hybridization at 42.degree. C. in
5.times.SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured
salmon sperm DNA, and 25% formamide, following standard Southern
blotting procedures for 12 to 24 hours. The carrier material is
finally washed three times each for 15 minutes using 0.2.times.SSC,
0.2% SDS at 45.degree. C.
[0085] Wild-type cellobiohydrolase: The term "wild-type"
cellobiohydrolase means a cellobiohydrolase naturally produced by a
microorganism, such as a bacterium, yeast, or filamentous fungus
found in nature.
[0086] Xylan-containing material: The term "xylan-containing
material" means any material comprising a plant cell wall
polysaccharide containing a backbone of beta-(1-4)-linked xylose
residues. Xylans of terrestrial plants are heteropolymers
possessing a beta-(1-4)-D-xylopyranose backbone, which is branched
by short carbohydrate chains. They comprise D-glucuronic acid or
its 4-O-methyl ether, L-arabinose, and/or various oligosaccharides,
composed of D-xylose, L-arabinose, D- or L-galactose, and
D-glucose. Xylan-type polysaccharides can be divided into
homoxylans and heteroxylans, which include glucuronoxylans,
(arabino)glucuronoxylans, (glucurono)arabinoxylans, arabinoxylans,
and complex heteroxylans. See, for example, Ebringerova et al.,
2005, Adv. Polym. Sci. 186: 1-67.
[0087] In the processes of the present invention, any material
containing xylan may be used. In a preferred aspect, the
xylan-containing material is lignocellulose.
[0088] Xylan degrading activity or xylanolytic activity: The term
"xylan degrading activity" or "xylanolytic activity" means a
biological activity that hydrolyzes xylan-containing material. The
two basic approaches for measuring xylanolytic activity include:
(1) measuring the total xylanolytic activity, and (2) measuring the
individual xylanolytic activities (e.g., endoxylanases,
beta-xylosidases, arabinofuranosidases, alpha-glucuronidases,
acetylxylan esterases, feruloyl esterases, and alpha-glucuronyl
esterases). Recent progress in assays of xylanolytic enzymes was
summarized in several publications including Biely and Puchard,
2006, Journal of the Science of Food and Agriculture 86(11):
1636-1647; Spanikova and Biely, 2006, FEBS Letters 580(19):
4597-4601; Herrimann et al., 1997, Biochemical Journal 321:
375-381.
[0089] Total xylan degrading activity can be measured by
determining the reducing sugars formed from various types of xylan,
including, for example, oat spelt, beechwood, and larchwood xylans,
or by photometric determination of dyed xylan fragments released
from various covalently dyed xylans. A common total xylanolytic
activity assay is based on production of reducing sugars from
polymeric 4-O-methyl glucuronoxylan as described in Bailey et al.,
1992, Interlaboratory testing of methods for assay of xylanase
activity, Journal of Biotechnology 23(3): 257-270. Xylanase
activity can also be determined with 0.2% AZCL-arabinoxylan as
substrate in 0.01% TRITON.RTM. X-100 and 200 mM sodium phosphate pH
6 at 37.degree. C. One unit of xylanase activity is defined as 1.0
.mu.mole of azurine produced per minute at 37.degree. C., pH 6 from
0.2% AZCL-arabinoxylan as substrate in 200 mM sodium phosphate pH
6.
[0090] For purposes of the present invention, xylan degrading
activity is determined by measuring the increase in hydrolysis of
birchwood xylan (Sigma Chemical Co., Inc., St. Louis, Mo., USA) by
xylan-degrading enzyme(s) under the following typical conditions: 1
ml reactions, 5 mg/ml substrate (total solids), 5 mg of xylanolytic
protein/g of substrate, 50 mM sodium acetate pH 5, 50.degree. C.,
24 hours, sugar analysis using p-hydroxybenzoic acid hydrazide
(PHBAH) assay as described by Lever, 1972, Anal. Biochem 47:
273-279.
[0091] Xylanase: The term "xylanase" means a
1,4-beta-D-xylan-xylohydrolase (E.C. 3.2.1.8) that catalyzes the
endohydrolysis of 1,4-beta-D-xylosidic linkages in xylans. For
purposes of the present invention, xylanase activity is determined
with 0.2% AZCL-arabinoxylan as substrate in 0.01% TRITON.RTM. X-100
and 200 mM sodium phosphate pH 6 at 37.degree. C. One unit of
xylanase activity is defined as 1.0 .mu.mole of azurine produced
per minute at 37.degree. C., pH 6 from 0.2% AZCL-arabinoxylan as
substrate in 200 mM sodium phosphate pH 6.
Conventions for Designation of Variants
[0092] For purposes of the present invention, the sequence of SEQ
ID NO: 4 is used to determine the corresponding amino acid residue
in another cellobiohydrolase. The amino acid sequence of another
cellobiohydrolase is aligned with SEQ ID NO: 4, and based on the
alignment, the amino acid position number corresponding to any
amino acid residue in SEQ ID NO: 4 is determined using the
Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol.
Biol. 48: 443-453) as implemented in the Needle program of the
EMBOSS package (EMBOSS: The European Molecular Biology Open
Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277),
preferably version 5.0.0 or later. The parameters used are a gap
open penalty of 10, a gap extension penalty of 0.5, and the
EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.
Numbering of the amino acid positions is based on the full-length
polypeptide (e.g., including the signal peptide) of SEQ ID NO: 4
wherein position -26 is the first amino acid of the signal peptide
(i.e., Met) and position 1 is Gln of SEQ ID NO: 4. For example,
based on the alignment, the position corresponding to position 265
of the Aspergillus fumigatus cellobiohydrolase I (SEQ ID NO: 4)
corresponds to position 265 of the Trichoderma reesei
cellobiohydrolase I (SEQ ID NO: 6) and position 262 of the
Rasamsonia emersonii cellobiohydrolase I (SEQ ID NO: 10).
[0093] Identification of the corresponding amino acid residue in
another cellobiohydrolase can be determined by an alignment of
multiple polypeptide sequences using several computer programs
including, but not limited to, MUSCLE (multiple sequence comparison
by log-expectation; version 3.5 or later; Edgar, 2004, Nucleic
Acids Research 32: 1792-1797), MAFFT (version 6.857 or later; Katoh
and Kuma, 2002, Nucleic Acids Research 30: 3059-3066; Katoh et al.,
2005, Nucleic Acids Research 33: 511-518; Katoh and Toh, 2007,
Bioinformatics 23: 372-374; Katoh et al., 2009, Methods in
Molecular Biology 537: 39-64; Katoh and Toh, 2010, Bioinformatics
26: 1899-1900), and EMBOSS EMMA employing ClustalW (1.83 or later;
Thompson et al., 1994, Nucleic Acids Research 22: 4673-4680), using
their respective default parameters.
[0094] When another cellobiohydrolase has diverged from the
referenced parent sequence (e.g., SEQ ID NO: 4) such that
traditional sequence-based comparison fails to detect their
relationship (Lindahl and Elofsson, 2000, J. Mol. Biol. 295:
613-615), other pairwise sequence comparison algorithms can be
used. Greater sensitivity in sequence-based searching can be
attained using search programs that utilize probabilistic
representations of polypeptide families (profiles) to search
databases. For example, the PSI-BLAST program generates profiles
through an iterative database search process and is capable of
detecting remote homologs (Atschul et al., 1997, Nucleic Acids Res.
25: 3389-3402). Even greater sensitivity can be achieved if the
family or superfamily for the polypeptide has one or more
representatives in the protein structure databases. Programs such
as GenTHREADER (Jones, 1999, J. Mol. Biol. 287: 797-815; McGuffin
and Jones, 2003, Bioinformatics 19: 874-881) utilize information
from a variety of sources (PSI-BLAST, secondary structure
prediction, structural alignment profiles, and solvation
potentials) as input to a neural network that predicts the
structural fold for a query sequence. Similarly, the method of
Gough et al., 2000, J. Mol. Biol. 313: 903-919, can be used to
align a sequence of unknown structure with the superfamily models
present in the SCOP database. These alignments can in turn be used
to generate homology models for the polypeptide, and such models
can be assessed for accuracy using a variety of tools developed for
that purpose.
[0095] For proteins of known structure, several tools and resources
are available for retrieving and generating structural alignments.
For example the SCOP superfamilies of proteins have been
structurally aligned, and those alignments are accessible and
downloadable. Two or more protein structures can be aligned using a
variety of algorithms such as the distance alignment matrix (Holm
and Sander, 1998, Proteins 33: 88-96) or combinatorial extension
(Shindyalov and Bourne, 1998, Protein Engineering 11: 739-747), and
implementation of these algorithms can additionally be utilized to
query structure databases with a structure of interest in order to
discover possible structural homologs (e.g., Holm and Park, 2000,
Bioinformatics 16: 566-567).
[0096] In describing the cellobiohydrolase variants of the present
invention, the nomenclature described below is adapted for ease of
reference. The accepted IUPAC single letter or three letter amino
acid abbreviation is employed.
[0097] Substitutions.
[0098] For an amino acid substitution, the following nomenclature
is used:
[0099] Original amino acid, position, substituted amino acid.
Accordingly, the substitution of threonine at position 226 with
alanine is designated as "Thr226Ala" or "T226A". Multiple mutations
are separated by addition marks ("+"), e.g., "Gly205Arg+Ser411Phe"
or "G205R+S411F", representing substitutions at positions 205 and
411 of glycine (G) with arginine (R) and serine (S) with
phenylalanine (F), respectively.
[0100] Deletions.
[0101] For an amino acid deletion, the following nomenclature is
used: Original amino acid, position, *. Accordingly, the deletion
of glycine at position 195 is designated as "Gly195*" or "G195*".
Multiple deletions are separated by addition marks ("+"), e.g.,
"Gly195*+Ser411*" or "G195*+S411*".
[0102] Insertions.
[0103] For an amino acid insertion, the following nomenclature is
used: Original amino acid, position, original amino acid, inserted
amino acid. Accordingly the insertion of lysine after glycine at
position 195 is designated "Gly195GlyLys" or "G195GK". An insertion
of multiple amino acids is designated [Original amino acid,
position, original amino acid, inserted amino acid #1, inserted
amino acid #2; etc.]. For example, the insertion of lysine and
alanine after glycine at position 195 is indicated as
"Gly195GlyLysAla" or "G195GKA".
[0104] In such cases the inserted amino acid residue(s) are
numbered by the addition of lower case letters to the position
number of the amino acid residue preceding the inserted amino acid
residue(s). In the above example, the sequence would thus be:
TABLE-US-00001 Parent: Variant: 195 195 195a 195b G G-K-A
[0105] Multiple Alterations.
[0106] Variants comprising multiple alterations are separated by
addition marks ("+"), e.g., "Arg170Tyr+Gly195Glu" or "R170Y+G195E"
representing a substitution of arginine and glycine at positions
170 and 195 with tyrosine and glutamic acid, respectively.
[0107] Different Alterations.
[0108] Where different alterations can be introduced at a position,
the different alterations are separated by a comma, e.g.,
"Arg170Tyr,Glu" represents a substitution of arginine at position
170 with tyrosine or glutamic acid. Thus, "Tyr167Gly,Ala+
Arg170Gly,Ala" designates the following variants: "Tyr167Gly+
Arg170Gly", "Tyr167Gly+ Arg170Ala", "Tyr167Ala+ Arg170Gly", and
"Tyr167Ala+Arg170Ala".
DETAILED DESCRIPTION OF THE INVENTION
[0109] The present invention relates to isolated cellobiohydrolase
variants, comprising an alteration at one or more (e.g., several)
positions corresponding to positions 4, 44, 45, 72, 265, 266, 391,
393 and 394 of SEQ ID NO: 4, wherein the variants have
cellobiohydrolase activity.
Variants
[0110] In an embodiment, the variant has a sequence identity of at
least 60%, e.g., at least 65%, at least 70%, at least 75%, at least
80%, at least 81%, at least 82%, at least 83%, at least 84%, at
least 85%, at least 86%, at least 87%, at least 88%, at least 89%,
at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, or at
least 99%, but less than 100%, to the amino acid sequence of the
parent cellobiohydrolase or the mature polypeptide thereof.
[0111] In another embodiment, the variant comprises a catalytic
domain having at least 60%, e.g., at least 65%, at least 70%, at
least 75%, at least 80%, at least 81%, at least 82%, at least 83%,
at least 84%, at least 85%, at least 86%, at least 87%, at least
88%, at least 89%, at least 90%, at least 95%, at least 96%, at
least 97%, at least 98%, or at least 99%, but less than 100%,
sequence identity to the catalytic domain of a parent
cellobiohydrolase.
[0112] In another embodiment, the variant has at least 60%, e.g.,
at least 65%, at least 70%, at least 75%, at least 80%, at least
81%, at least 82%, at least 83%, at least 84%, at least 85%, at
least 86%, at least 87%, at least 88%, at least 89%, at least 90%,
at least 91%, at least 92%, at least 93%, at least 94%, at least
95%, at least 96%, at least 97%, at least 98%, or at least 99%, but
less than 100%, sequence identity to the mature polypeptide of SEQ
ID NO: 4. In another embodiment, the variant comprises a catalytic
domain having at least 60%, e.g., at least 65%, at least 70%, at
least 75%, at least 80%, at least 81%, at least 82%, at least 83%,
at least 84%, at least 85%, at least 86%, at least 87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, or at least 99%, but less than 100%, sequence
identity to amino acids 1 to 437 of SEQ ID NO: 4.
[0113] In another embodiment, the variant has at least 60%, e.g.,
at least 65%, at least 70%, at least 75%, at least 80%, at least
81%, at least 82%, at least 83%, at least 84%, at least 85%, at
least 86%, at least 87%, at least 88%, at least 89%, at least 90%,
at least 91%, at least 92%, at least 93%, at least 94%, at least
95%, at least 96%, at least 97%, at least 98%, or at least 99%, but
less than 100%, sequence identity to the mature polypeptide of SEQ
ID NO: 6. In another embodiment, the variant comprises a catalytic
domain having at least 60%, e.g., at least 65%, at least 70%, at
least 75%, at least 80%, at least 81%, at least 82%, at least 83%,
at least 84%, at least 85%, at least 86%, at least 87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, or at least 99%, but less than 100%, sequence
identity to amino acids 1 to 429 of SEQ ID NO: 6.
[0114] In another embodiment, the variant has at least 60%, e.g.,
at least 65%, at least 70%, at least 75%, at least 80%, at least
81%, at least 82%, at least 83%, at least 84%, at least 85%, at
least 86%, at least 87%, at least 88%, at least 89%, at least 90%,
at least 91%, at least 92%, at least 93%, at least 94%, at least
95%, at least 96%, at least 97%, at least 98%, or at least 99%, but
less than 100%, sequence identity to the mature polypeptide of SEQ
ID NO: 8. In another embodiment, the variant comprises a catalytic
domain having at least 60%, e.g., at least 65%, at least 70%, at
least 75%, at least 80%, at least 81%, at least 82%, at least 83%,
at least 84%, at least 85%, at least 86%, at least 87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, or at least 99%, but less than 100%, sequence
identity to amino acids 1 to 440 of SEQ ID NO: 8.
[0115] In another embodiment, the variant has at least 60%, e.g.,
at least 65%, at least 70%, at least 75%, at least 80%, at least
81%, at least 82%, at least 83%, at least 84%, at least 85%, at
least 86%, at least 87%, at least 88%, at least 89%, at least 90%,
at least 91%, at least 92%, at least 93%, at least 94%, at least
95%, at least 96%, at least 97%, at least 98%, or at least 99%, but
less than 100%, sequence identity to the mature polypeptide of SEQ
ID NO: 10. In another embodiment, the variant comprises a catalytic
domain having at least 60%, e.g., at least 65%, at least 70%, at
least 75%, at least 80%, at least 81%, at least 82%, at least 83%,
at least 84%, at least 85%, at least 86%, at least 87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, or at least 99%, but less than 100%, sequence
identity to amino acids 1 to 437 of SEQ ID NO: 10.
[0116] In another embodiment, the variant has at least 60%, e.g.,
at least 65%, at least 70%, at least 75%, at least 80%, at least
81%, at least 82%, at least 83%, at least 84%, at least 85%, at
least 86%, at least 87%, at least 88%, at least 89%, at least 90%,
at least 91%, at least 92%, at least 93%, at least 94%, at least
95%, at least 96%, at least 97%, at least 98%, or at least 99%, but
less than 100%, sequence identity to the mature polypeptide of SEQ
ID NO: 12. In another embodiment, the variant comprises a catalytic
domain having at least 60%, e.g., at least 65%, at least 70%, at
least 75%, at least 80%, at least 81%, at least 82%, at least 83%,
at least 84%, at least 85%, at least 86%, at least 87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, or at least 99%, but less than 100%, sequence
identity to amino acids 1 to 437 of SEQ ID NO: 12.
[0117] In another embodiment, the variant has at least 60%, e.g.,
at least 65%, at least 70%, at least 75%, at least 80%, at least
81%, at least 82%, at least 83%, at least 84%, at least 85%, at
least 86%, at least 87%, at least 88%, at least 89%, at least 90%,
at least 91%, at least 92%, at least 93%, at least 94%, at least
95%, at least 96%, at least 97%, at least 98%, or at least 99%, but
less than 100%, sequence identity to the mature polypeptide of SEQ
ID NO: 14. In another embodiment, the variant comprises a catalytic
domain having at least 60%, e.g., at least 65%, at least 70%, at
least 75%, at least 80%, at least 81%, at least 82%, at least 83%,
at least 84%, at least 85%, at least 86%, at least 87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, or at least 99%, but less than 100%, sequence
identity to amino acids 1 to 438 of SEQ ID NO: 14.
[0118] In another embodiment, the variant has at least 60%, e.g.,
at least 65%, at least 70%, at least 75%, at least 80%, at least
81%, at least 82%, at least 83%, at least 84%, at least 85%, at
least 86%, at least 87%, at least 88%, at least 89%, at least 90%,
at least 91%, at least 92%, at least 93%, at least 94%, at least
95%, at least 96%, at least 97%, at least 98%, or at least 99%, but
less than 100%, sequence identity to the mature polypeptide of SEQ
ID NO: 16. In another embodiment, the variant comprises a catalytic
domain having at least 60%, e.g., at least 65%, at least 70%, at
least 75%, at least 80%, at least 81%, at least 82%, at least 83%,
at least 84%, at least 85%, at least 86%, at least 87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, or at least 99%, but less than 100%, sequence
identity to amino acids 1 to 437 of SEQ ID NO: 16.
[0119] In another embodiment, the variant has at least 60%, e.g.,
at least 65%, at least 70%, at least 75%, at least 80%, at least
81%, at least 82%, at least 83%, at least 84%, at least 85%, at
least 86%, at least 87%, at least 88%, at least 89%, at least 90%,
at least 91%, at least 92%, at least 93%, at least 94%, at least
95%, at least 96%, at least 97%, at least 98%, or at least 99%, but
less than 100%, sequence identity to the mature polypeptide of SEQ
ID NO: 18. In another embodiment, the variant comprises a catalytic
domain having at least 60%, e.g., at least 65%, at least 70%, at
least 75%, at least 80%, at least 81%, at least 82%, at least 83%,
at least 84%, at least 85%, at least 86%, at least 87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, or at least 99%, but less than 100%, sequence
identity to amino acids 1 to 430 of SEQ ID NO: 18.
[0120] In another embodiment, the variant has at least 60%, e.g.,
at least 65%, at least 70%, at least 75%, at least 80%, at least
81%, at least 82%, at least 83%, at least 84%, at least 85%, at
least 86%, at least 87%, at least 88%, at least 89%, at least 90%,
at least 91%, at least 92%, at least 93%, at least 94%, at least
95%, at least 96%, at least 97%, at least 98%, or at least 99%, but
less than 100%, sequence identity to the mature polypeptide of SEQ
ID NO: 20. In another embodiment, the variant comprises a catalytic
domain having at least 60%, e.g., at least 65%, at least 70%, at
least 75%, at least 80%, at least 81%, at least 82%, at least 83%,
at least 84%, at least 85%, at least 86%, at least 87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, or at least 99%, but less than 100%, sequence
identity to amino acids 1 to 433 of SEQ ID NO: 20.
[0121] In another embodiment, the variant has at least 60%, e.g.,
at least 65%, at least 70%, at least 75%, at least 80%, at least
81%, at least 82%, at least 83%, at least 84%, at least 85%, at
least 86%, at least 87%, at least 88%, at least 89%, at least 90%,
at least 91%, at least 92%, at least 93%, at least 94%, at least
95%, at least 96%, at least 97%, at least 98%, or at least 99%, but
less than 100%, sequence identity to the mature polypeptide of SEQ
ID NO: 22. In another embodiment, the variant comprises a catalytic
domain having at least 60%, e.g., at least 65%, at least 70%, at
least 75%, at least 80%, at least 81%, at least 82%, at least 83%,
at least 84%, at least 85%, at least 86%, at least 87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, or at least 99%, but less than 100%, sequence
identity to amino acids 1 to 438 of SEQ ID NO: 22.
[0122] In one aspect, the number of alterations in the variants of
the present invention is 1-9, e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9
alterations. In another aspect, the number of substitutions in the
variants of the present invention is 1-2, e.g., 1 or 2
substitutions.
[0123] In another aspect, the variant comprises an alteration at
one or more (e.g., several) positions corresponding to positions 4,
44, 45, 72, 265, 266, 391, 393 and 394 of SEQ ID NO: 4, wherein the
alteration is a substitution. In another aspect, a variant
comprises an alteration at two positions corresponding to any of
positions 4, 44, 45, 72, 265, 266, 391, 393 and 394. In another
aspect, a variant comprises an alteration at three positions
corresponding to any of positions 4, 44, 45, 72, 265, 266, 391, 393
and 394. In another aspect, a variant comprises an alteration at
four positions corresponding to any of positions 4, 44, 45, 72,
265, 266, 391, 393 and 394. In another aspect, a variant comprises
an alteration at five positions corresponding to any of positions
4, 44, 45, 72, 265, 266, 391, 393 and 394. In another aspect, a
variant comprises an alteration at six positions corresponding to
any of positions 4, 44, 45, 72, 265, 266, 391, 393 and 394. In
another aspect, a variant comprises an alteration at seven
positions corresponding to any of positions 4, 44, 45, 72, 265,
266, 391, 393 and 394. In another aspect, a variant comprises an
alteration at eight positions corresponding to any of positions 4,
44, 45, 72, 265, 266, 391, 393 and 394. In another aspect, a
variant comprises an alteration at each position corresponding to
positions 4, 44, 45, 72, 265, 266, 391, 393 and 394.
[0124] In another aspect, the variant comprises or consists of a
substitution at a position corresponding to position 4 of SEQ ID
NO: 4. In another aspect, the amino acid at a position
corresponding to position 4 is substituted with Ala, Arg, Asn, Asp,
Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,
Trp, Tyr, or Val, e.g., with Cys. In another aspect, the variant
comprises or consists of the substitution of Gly Cys at a position
corresponding to position 4 of SEQ ID NO: 4 (e.g., G4C).
[0125] In another aspect, the variant comprises or consists of a
substitution at a position corresponding to position 44 of SEQ ID
NO: 4. In another aspect, the amino acid at a position
corresponding to position 44 is substituted with Ala, Arg, Asn,
Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,
Thr, Trp, Tyr, or Val, e.g., with Ser, Thr, Ala, Gly, Ile, Met,
Asn, or Lys. In another aspect, the variant comprises or consists
of the substitution of Val.fwdarw.Ser, Val.fwdarw.Thr,
Val.fwdarw.Ala, Val.fwdarw.Gly, Val.fwdarw.Ile, Val.fwdarw.Met,
Val.fwdarw.Asn, or Val.fwdarw.Lys at a position corresponding to
position 44 of SEQ ID NO: 4 (e.g., V44S, V44T, V44A, V44G, V44I,
V44M, V44N, or V44K).
[0126] In another aspect, the variant comprises or consists of a
substitution at a position corresponding to position 45 of SEQ ID
NO: 4. In another aspect, the amino acid at a position
corresponding to position 45 is substituted with Ala, Arg, Asn,
Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,
Thr, Trp, Tyr, or Val, e.g., with Ser or Asn. In another aspect,
the variant comprises or consists of the substitution of
Gly.fwdarw.Ser or Gly.fwdarw.Asn at a position corresponding to
position 45 of SEQ ID NO: 4 (e.g., G45S, G45N).
[0127] In another aspect, the variant comprises or consists of a
substitution at a position corresponding to position 72 of SEQ ID
NO: 4. In another aspect, the amino acid at a position
corresponding to position 72 is substituted with Ala, Arg, Asn,
Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,
Thr, Trp, Tyr, or Val, e.g., with Cys. In another aspect, the
variant comprises or consists of the substitution of Ala.fwdarw.Cys
at a position corresponding to position 72 of SEQ ID NO: 4 (e.g.,
A72C).
[0128] In another aspect, the variant comprises or consists of
substitutions at a positions corresponding to positions 4 and 72 of
SEQ ID NO: 4, e.g., with two cysteine residues that may form a
disulfide bond. In another aspect, the amino acid at a position
corresponding to position 4 is substituted with a Cys and the amino
acid at a position corresponding to position 72 is substituted with
a Cys. In another aspect, the variant comprises or consists of the
substitutions of Gly.fwdarw.Cys at a position corresponding to
position 4 of SEQ ID NO: 4 (e.g., G4C) and Ala.fwdarw.Cys at a
position corresponding to position 72 of SEQ ID NO: 4 (e.g.,
A72C).
[0129] In another aspect, the variant comprises or consists of a
substitution at a position corresponding to position 265 of SEQ ID
NO: 4. In another aspect, the amino acid at a position
corresponding to position 44 is substituted with Ala, Arg, Asn,
Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,
Thr, Trp, Tyr, or Val, e.g., with Gly, Pro, or Ala. In another
aspect, the variant comprises or consists of the substitution of
Ser.fwdarw.Cys, Ser.fwdarw.Gly, Ser.fwdarw.Pro, or Ser.fwdarw.Ala
at a position corresponding to position 265 of SEQ ID NO: 4 (e.g.,
S265C, S265G, S265P, S265A).
[0130] In another aspect, the variant comprises or consists of a
substitution at a position corresponding to position 266 of SEQ ID
NO: 4. In another aspect, the amino acid at a position
corresponding to position 266 is substituted with Ala, Arg, Asn,
Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,
Thr, Trp, Tyr, or Val, e.g., with Tyr. In another aspect, the
variant comprises or consists of the substitution of Phe.fwdarw.Tyr
at a position corresponding to position 266 of SEQ ID NO: 4 (e.g.,
F266Y).
[0131] In another aspect, the variant comprises or consists of a
substitution at a position corresponding to position 391 of SEQ ID
NO: 4. In another aspect, the amino acid at a position
corresponding to position 391 is substituted with Ala, Arg, Asn,
Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,
Thr, Trp, Tyr, or Val, e.g., with Tyr, Trp, Val, or Asn. In another
aspect, the variant comprises or consists of the substitution of
Thr.fwdarw.Asp, Thr.fwdarw.Trp, Thr.fwdarw.Val, or Thr.fwdarw.Asn
at a position corresponding to position 391 of SEQ ID NO: 4 (e.g.,
T391D, T391W, T391V, or T391N).
[0132] In another aspect, the variant comprises or consists of a
substitution at a position corresponding to position 393 of SEQ ID
NO: 4. In another aspect, the amino acid at a position
corresponding to position 393 is substituted with Ala, Arg, Asn,
Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,
Thr, Trp, Tyr, or Val, e.g., with Tyr. In another aspect, the
variant comprises or consists of the substitution of Ser.fwdarw.Asp
at a position corresponding to position 393 of SEQ ID NO: 4 (e.g.,
S393D).
[0133] In another aspect, the variant comprises or consists of a
substitution at a position corresponding to position 394 of SEQ ID
NO: 4. In another aspect, the amino acid at a position
corresponding to position 394 is substituted with Ala, Arg, Asn,
Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,
Thr, Trp, Tyr, or Val, e.g., with Tyr. In another aspect, the
variant comprises or consists of the substitution of Ser.fwdarw.Pro
at a position corresponding to position 394 of SEQ ID NO: 4 (e.g.,
S394P).
[0134] In another embodiment, the variant comprises or consists of
SEQ ID NO: 26 or the mature polypeptide thereof. In one embodiment,
the variant comprises a catalytic domain comprising amino acids 1
to 437 of SEQ ID NO: 26.
[0135] In another embodiment, the variant comprises or consists of
SEQ ID NO: 28 or the mature polypeptide thereof. In one embodiment,
the variant comprises a catalytic domain comprising amino acids 1
to 437 of SEQ ID NO: 28.
[0136] In another embodiment, the variant comprises or consists of
SEQ ID NO: 38 or the mature polypeptide thereof. In one embodiment,
the variant comprises a catalytic domain comprising amino acids 1
to 437 of SEQ ID NO: 38.
[0137] In another embodiment, the variant comprises or consists of
SEQ ID NO: 42 or the mature polypeptide thereof. In one embodiment,
the variant comprises a catalytic domain comprising amino acids 1
to 437 of SEQ ID NO: 42.
[0138] In another embodiment, the variant comprises or consists of
SEQ ID NO: 46 or the mature polypeptide thereof. In one embodiment,
the variant comprises a catalytic domain comprising amino acids 1
to 437 of SEQ ID NO: 46.
[0139] In another embodiment, the variant comprises or consists of
SEQ ID NO: 48 or the mature polypeptide thereof. In one embodiment,
the variant comprises a catalytic domain comprising amino acids 1
to 437 of SEQ ID NO: 48.
[0140] In another embodiment, the variant comprises or consists of
SEQ ID NO: 67 or the mature polypeptide thereof. In one embodiment,
the variant comprises a catalytic domain comprising amino acids 1
to 437 of SEQ ID NO: 67.
[0141] In another embodiment, the variant comprises or consists of
SEQ ID NO: 69 or the mature polypeptide thereof. In one embodiment,
the variant comprises a catalytic domain comprising amino acids 1
to 437 of SEQ ID NO: 69.
[0142] In another embodiment, the variant comprises or consists of
SEQ ID NO: 71 or the mature polypeptide thereof. In one embodiment,
the variant comprises a catalytic domain comprising amino acids 1
to 437 of SEQ ID NO: 71.
[0143] In another embodiment, the variant comprises or consists of
SEQ ID NO: 73 or the mature polypeptide thereof. In one embodiment,
the variant comprises a catalytic domain comprising amino acids 1
to 437 of SEQ ID NO: 73.
[0144] In another embodiment, the variant comprises or consists of
SEQ ID NO: 75 or the mature polypeptide thereof. In one embodiment,
the variant comprises a catalytic domain comprising amino acids 1
to 437 of SEQ ID NO: 75.
[0145] In another embodiment, the variant comprises or consists of
SEQ ID NO: 77 or the mature polypeptide thereof. In one embodiment,
the variant comprises a catalytic domain comprising amino acids 1
to 437 of SEQ ID NO: 77.
[0146] In another embodiment, the variant comprises or consists of
SEQ ID NO: 79 or the mature polypeptide thereof. In one embodiment,
the variant comprises a catalytic domain comprising amino acids 1
to 437 of SEQ ID NO: 79.
[0147] In another embodiment, the variant comprises or consists of
SEQ ID NO: 81 or the mature polypeptide thereof. In one embodiment,
the variant comprises a catalytic domain comprising amino acids 1
to 437 of SEQ ID NO: 81.
[0148] In another embodiment, the variant comprises or consists of
SEQ ID NO: 83 or the mature polypeptide thereof. In one embodiment,
the variant comprises a catalytic domain comprising amino acids 1
to 437 of SEQ ID NO: 83.
[0149] In another embodiment, the variant comprises or consists of
SEQ ID NO: 85 or the mature polypeptide thereof. In one embodiment,
the variant comprises a catalytic domain comprising amino acids 1
to 437 of SEQ ID NO: 85.
[0150] The variants may further comprise one or more additional
alterations, e.g., substitutions, insertions, or deletions at one
or more (e.g., several) other positions, such as a substitution, a
deletion, and/or an insertion at one or more (e.g., several) other
positions disclosed in WO 2014/138672, WO 2011/050037, WO
2005/028636, WO 2005/001065, WO 2004/016760, U.S. Pat. No.
7,375,197, and U.S. Provisional Application No. 62/046,344 filed
Sep. 5, 2014, and U.S. Provisional Application No. 62/047,296,
filed Sep. 8, 2014; which are incorporated herein in their
entireties.
[0151] The amino acid changes may be of a minor nature, that is
conservative amino acid substitutions or insertions that do not
significantly affect the folding and/or activity of the protein;
small deletions, typically of 1-30 amino acids; small amino- or
carboxyl-terminal extensions, such as an amino-terminal methionine
residue; a small linker peptide of up to 20-25 residues; or a small
extension that facilitates purification by changing net charge or
another function, such as a poly-histidine tract, an antigenic
epitope or a binding domain.
[0152] Examples of conservative substitutions are within the groups
of basic amino acids (arginine, lysine and histidine), acidic amino
acids (glutamic acid and aspartic acid), polar amino acids
(glutamine and asparagine), hydrophobic amino acids (leucine,
isoleucine and valine), aromatic amino acids (phenylalanine,
tryptophan and tyrosine), and small amino acids (glycine, alanine,
serine, threonine and methionine). Amino acid substitutions that do
not generally alter specific activity are known in the art and are
described, for example, by H. Neurath and R. L. Hill, 1979, In, The
Proteins, Academic Press, New York. Common substitutions are
Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn,
Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile,
Leu/Val, Ala/Glu, and Asp/Gly.
[0153] Alternatively, the amino acid changes are of such a nature
that the physico-chemical properties of the polypeptides are
altered. For example, amino acid changes may improve the thermal
stability of the polypeptide, alter the substrate specificity,
change the pH optimum, and the like.
[0154] Essential amino acids in a polypeptide can be identified
according to procedures known in the art, such as site-directed
mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells,
1989, Science 244: 1081-1085). In the latter technique, single
alanine mutations are introduced at every residue in the molecule,
and the resultant mutant molecules are tested for cellobiohydrolase
activity to identify amino acid residues that are critical to the
activity of the molecule. See also, Hilton et al., 1996, J. Biol.
Chem. 271: 4699-4708. The active site of the enzyme or other
biological interaction can also be determined by physical analysis
of structure, as determined by such techniques as nuclear magnetic
resonance, crystallography, electron diffraction, or photoaffinity
labeling, in conjunction with mutation of putative contact site
amino acids. See, for example, de Vos et al., 1992, Science 255:
306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver
et al., 1992, FEBS Lett. 309: 59-64. The identity of essential
amino acids can also be inferred from an alignment with a related
polypeptide.
[0155] Additional guidance on the structure-activity relationship
of the variants herein can be determined using published
three-dimensional structure data for the Cel7A family of
cellobiohydrolases (e.g., See, Moroz et al., 2015, Acta Cryst. F71:
114-120).
[0156] In each of the embodiments described above, a variant of the
present invention may be a hybrid polypeptide (chimera) in which a
region of the variant is replaced with a region of another
polypeptide. In one aspect, the region is a carbohydrate binding
domain. The carbohydrate binding domain of a variant may be
replaced with another (heterologous) carbohydrate binding
domain.
[0157] In each of the embodiments described above, a variant of the
present invention may be a fusion polypeptide or cleavable fusion
polypeptide in which another polypeptide is fused at the N-terminus
or the C-terminus of the variant. In one aspect, the other
polypeptide is a carbohydrate binding domain. The catalytic domain
of a variant of the present invention without a carbohydrate
binding domain may be fused to a carbohydrate binding domain. A
fusion polypeptide is produced by fusing a polynucleotide encoding
another polypeptide to a polynucleotide encoding a variant of the
present invention. Techniques for producing fusion polypeptides are
known in the art, and include ligating the coding sequences
encoding the polypeptides so that they are in frame and expression
of the fusion polypeptide is under control of the same promoter(s)
and terminator. Fusion polypeptides may also be constructed using
intein technology in which fusion polypeptides are created
post-translationally (Cooper et al., 1993, EMBO J. 12: 2575-2583;
Dawson et al., 1994, Science 266: 776-779).
[0158] In one embodiment, the variant is a hybrid or chimeric
polypeptide in which the carbohydrate binding domain of the variant
is replaced with a different carbohydrate binding domain. In
another embodiment, the variant is a fusion protein in which a
heterologous carbohydrate binding domain is fused to the variant.
For example, the variant may comprise a variant Rasamsonia
emersonii cellobiohydrolase catalytic domain fused to the
Trichoderma reesei carbohydrate binding domain. Accordingly, in one
embodiment, the variant has at least 60%, e.g., at least 65%, at
least 70%, at least 75%, at least 80%, at least 81%, at least 82%,
at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at least 88%, at least 89%, at least 90%, at least 91%, at
least 92%, at least 93%, at least 94%, at least 95%, at least 96%,
at least 97%, at least 98%, or at least 99%, but less than 100%,
sequence identity to the mature polypeptide of SEQ ID NO: 24. In
one aspect, the carbohydrate binding domain is fused to the
N-terminus of the variant. In another aspect, the carbohydrate
binding domain is fused to the C-terminus of the variant.
[0159] In another embodiment, the variant comprises or consists of
SEQ ID NO: 63 or the mature polypeptide thereof. In one embodiment,
the variant comprises a catalytic domain comprising amino acids 1
to 437 of SEQ ID NO: 63.
[0160] A fusion polypeptide can further comprise a cleavage site
between the two polypeptides. Upon secretion of the fusion protein,
the site is cleaved releasing the two polypeptides. Examples of
cleavage sites include, but are not limited to, the sites disclosed
in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3: 568-576;
Svetina et al., 2000, J. Biotechnol. 76: 245-251; Rasmussen-Wilson
et al., 1997, Appl. Environ. Microbiol. 63: 3488-3493; Ward et al.,
1995, Biotechnology 13: 498-503; and Contreras et al., 1991,
Biotechnology 9: 378-381; Eaton et al., 1986, Biochemistry 25:
505-512; Collins-Racie et al., 1995, Biotechnology 13: 982-987;
Carter et al., 1989, Proteins: Structure, Function, and Genetics 6:
240-248; and Stevens, 2003, Drug Discovery World 4: 35-48.
[0161] In an embodiment, the variant has increased specific
performance compared to the parent enzyme.
Parent Cellobiohydrolases
[0162] The parent cellobiohydrolase may be any cellobiohydrolase
I.
[0163] In one embodiment, the parent cellobiohydrolase may be: (a)
a polypeptide having at least 60% sequence identity to the mature
polypeptide of SEQ ID NO: 4; (b) a polypeptide encoded by a
polynucleotide that hybridizes under low stringency conditions with
(i) the mature polypeptide coding sequence of SEQ ID NO: 1; (ii)
the cDNA sequence thereof, or (iii) the full-length complement of
(i) or (ii); (c) a polypeptide encoded by a polynucleotide having
at least 60% identity to the mature polypeptide coding sequence of
SEQ ID NO: 1, or the cDNA sequence thereof; or (d) a fragment of
the mature polypeptide of SEQ ID NO: 4, which has cellobiohydrolase
activity.
[0164] In another embodiment, the parent cellobiohydrolase may also
be: (a) a polypeptide having at least 60% sequence identity to the
mature polypeptide of SEQ ID NO: 6, (b) a polypeptide encoded by a
polynucleotide that hybridizes under low stringency conditions with
(i) the mature polypeptide coding sequence of SEQ ID NO: 5, (ii)
the cDNA sequence thereof, or (iii) the full-length complement of
(i) or (ii); (c) a polypeptide encoded by a polynucleotide having
at least 60% identity to the mature polypeptide coding sequence of
SEQ ID NO: 5, or the cDNA sequence thereof; or (d) a fragment of
the mature polypeptide of SEQ ID NO: 6, which has cellobiohydrolase
activity.
[0165] In another embodiment, the parent cellobiohydrolase may also
be: (a) a polypeptide having at least 60% sequence identity to the
mature polypeptide of SEQ ID NO: 8, (b) a polypeptide encoded by a
polynucleotide that hybridizes under low stringency conditions with
(i) the mature polypeptide coding sequence of SEQ ID NO: 7, (ii)
the cDNA sequence thereof, or (iii) the full-length complement of
(i) or (ii); (c) a polypeptide encoded by a polynucleotide having
at least 60% identity to the mature polypeptide coding sequence of
SEQ ID NO: 7, or the cDNA sequence thereof; or (d) a fragment of
the mature polypeptide of SEQ ID NO: 8, which has cellobiohydrolase
activity.
[0166] In another embodiment, the parent cellobiohydrolase may also
be: (a) a polypeptide having at least 60% sequence identity to the
mature polypeptide of SEQ ID NO: 10, (b) a polypeptide encoded by a
polynucleotide that hybridizes under low stringency conditions with
(i) the mature polypeptide coding sequence of SEQ ID NO: 9, (ii)
the cDNA sequence thereof, or (iii) the full-length complement of
(i) or (ii); (c) a polypeptide encoded by a polynucleotide having
at least 60% identity to the mature polypeptide coding sequence of
SEQ ID NO: 9, or the cDNA sequence thereof; or (d) a fragment of
the mature polypeptide of SEQ ID NO: 10, which has
cellobiohydrolase activity.
[0167] In another embodiment, the parent cellobiohydrolase may also
be: (a) a polypeptide having at least 60% sequence identity to the
mature polypeptide of SEQ ID NO: 12, (b) a polypeptide encoded by a
polynucleotide that hybridizes under low stringency conditions with
(i) the mature polypeptide coding sequence of SEQ ID NO: 11, (ii)
the cDNA sequence thereof, or (iii) the full-length complement of
(i) or (ii); (c) a polypeptide encoded by a polynucleotide having
at least 60% identity to the mature polypeptide coding sequence of
SEQ ID NO: 11, or the cDNA sequence thereof; or (d) a fragment of
the mature polypeptide of SEQ ID NO: 12, which has
cellobiohydrolase activity.
[0168] In another embodiment, the parent cellobiohydrolase may also
be: (a) a polypeptide having at least 60% sequence identity to the
mature polypeptide of SEQ ID NO: 14, (b) a polypeptide encoded by a
polynucleotide that hybridizes under low stringency conditions with
(i) the mature polypeptide coding sequence of SEQ ID NO: 13, (ii)
the cDNA sequence thereof, or (iii) the full-length complement of
(i) or (ii); (c) a polypeptide encoded by a polynucleotide having
at least 60% identity to the mature polypeptide coding sequence of
SEQ ID NO: 13, or the cDNA sequence thereof; or (d) a fragment of
the mature polypeptide of SEQ ID NO: 14, which has
cellobiohydrolase activity.
[0169] In another embodiment, the parent cellobiohydrolase may also
be: (a) a polypeptide having at least 60% sequence identity to the
mature polypeptide of SEQ ID NO: 16, (b) a polypeptide encoded by a
polynucleotide that hybridizes under low stringency conditions with
(i) the mature polypeptide coding sequence of SEQ ID NO: 15, (ii)
the cDNA sequence thereof, or (iii) the full-length complement of
(i) or (ii); (c) a polypeptide encoded by a polynucleotide having
at least 60% identity to the mature polypeptide coding sequence of
SEQ ID NO: 15, or the cDNA sequence thereof; or (d) a fragment of
the mature polypeptide of SEQ ID NO: 16, which has
cellobiohydrolase activity.
[0170] In another embodiment, the parent cellobiohydrolase may also
be: (a) a polypeptide having at least 60% sequence identity to the
mature polypeptide of SEQ ID NO: 18, (b) a polypeptide encoded by a
polynucleotide that hybridizes under low stringency conditions with
(i) the mature polypeptide coding sequence of SEQ ID NO: 17, (ii)
the cDNA sequence thereof, or (iii) the full-length complement of
(i) or (ii); (c) a polypeptide encoded by a polynucleotide having
at least 60% identity to the mature polypeptide coding sequence of
SEQ ID NO: 17, or the cDNA sequence thereof; or (d) a fragment of
the mature polypeptide of SEQ ID NO: 18, which has
cellobiohydrolase activity.
[0171] In another embodiment, the parent cellobiohydrolase may also
be: (a) a polypeptide having at least 60% sequence identity to the
mature polypeptide of SEQ ID NO: 20, (b) a polypeptide encoded by a
polynucleotide that hybridizes under low stringency conditions with
(i) the mature polypeptide coding sequence of SEQ ID NO: 19, (ii)
the cDNA sequence thereof, or (iii) the full-length complement of
(i) or (ii); (c) a polypeptide encoded by a polynucleotide having
at least 60% identity to the mature polypeptide coding sequence of
SEQ ID NO: 19, or the cDNA sequence thereof; or (d) a fragment of
the mature polypeptide of SEQ ID NO: 20, which has
cellobiohydrolase activity.
[0172] In another embodiment, the parent cellobiohydrolase may also
be: (a) a polypeptide having at least 60% sequence identity to the
mature polypeptide of SEQ ID NO: 22, (b) a polypeptide encoded by a
polynucleotide that hybridizes under low stringency conditions with
(i) the mature polypeptide coding sequence of SEQ ID NO: 21, (ii)
the cDNA sequence thereof, or (iii) the full-length complement of
(i) or (ii); (c) a polypeptide encoded by a polynucleotide having
at least 60% identity to the mature polypeptide coding sequence of
SEQ ID NO: 21, or the cDNA sequence thereof; or (d) a fragment of
the mature polypeptide of SEQ ID NO: 22, which has
cellobiohydrolase activity.
[0173] In another embodiment, the parent cellobiohydrolase may also
be a fusion protein such as: (a) a polypeptide having at least 60%
sequence identity to the mature polypeptide of SEQ ID NO: 52, (b) a
polypeptide encoded by a polynucleotide that hybridizes under low
stringency conditions with (i) the mature polypeptide coding
sequence of SEQ ID NO: 51, (ii) the cDNA sequence thereof, or (iii)
the full-length complement of (i) or (ii); (c) a polypeptide
encoded by a polynucleotide having at least 60% identity to the
mature polypeptide coding sequence of SEQ ID NO: 51, or the cDNA
sequence thereof; or (d) a fragment of the mature polypeptide of
SEQ ID NO: 52, which has cellobiohydrolase activity.
[0174] In one aspect, the parent has a sequence identity to the
mature polypeptide of SEQ ID NO: 4 of at least 60%, e.g., at least
65%, at least 70%, at least 75%, at least 80%, at least 81%, at
least 82%, at least 83%, at least 84%, at least 85%, at least 86%,
at least 87%, at least 88%, at least 89%, at least 90%, at least
91%, at least 92%, at least 93%, at least 94%, at least 95%, at
least 96%, at least 97%, at least 98%, at least 99%, or 100%, which
have cellobiohydrolase activity. In another aspect, the amino acid
sequence of the parent differs by up to 10 amino acids, e.g., 1, 2,
3, 4, 5, 6, 7, 8, 9, or 10, from the mature polypeptide of SEQ ID
NO: 4.
[0175] In one aspect, the parent has a sequence identity to the
mature polypeptide of SEQ ID NO: 6 of at least 60%, e.g., at least
65%, at least 70%, at least 75%, at least 80%, at least 81%, at
least 82%, at least 83%, at least 84%, at least 85%, at least 86%,
at least 87%, at least 88%, at least 89%, at least 90%, at least
91%, at least 92%, at least 93%, at least 94%, at least 95%, at
least 96%, at least 97%, at least 98%, at least 99%, or 100%, which
have cellobiohydrolase activity. In another aspect, the amino acid
sequence of the parent differs by up to 10 amino acids, e.g., 1, 2,
3, 4, 5, 6, 7, 8, 9, or 10, from the mature polypeptide of SEQ ID
NO: 6.
[0176] In another aspect, the parent has a sequence identity to the
mature polypeptide of SEQ ID NO: 8 of at least 60%, e.g., at least
65%, at least 70%, at least 75%, at least 80%, at least 81%, at
least 82%, at least 83%, at least 84%, at least 85%, at least 86%,
at least 87%, at least 88%, at least 89%, at least 90%, at least
91%, at least 92%, at least 93%, at least 94%, at least 95%, at
least 96%, at least 97%, at least 98%, at least 99%, or 100%, which
have cellobiohydrolase activity. In another aspect, the amino acid
sequence of the parent differs by up to 10 amino acids, e.g., 1, 2,
3, 4, 5, 6, 7, 8, 9, or 10, from the mature polypeptide of SEQ ID
NO: 8.
[0177] In another aspect, the parent has a sequence identity to the
mature polypeptide of SEQ ID NO: 10 of at least 60%, e.g., at least
65%, at least 70%, at least 75%, at least 80%, at least 81%, at
least 82%, at least 83%, at least 84%, at least 85%, at least 86%,
at least 87%, at least 88%, at least 89%, at least 90%, at least
91%, at least 92%, at least 93%, at least 94%, at least 95%, at
least 96%, at least 97%, at least 98%, at least 99%, or 100%, which
have cellobiohydrolase activity. In another aspect, the amino acid
sequence of the parent differs by up to 10 amino acids, e.g., 1, 2,
3, 4, 5, 6, 7, 8, 9, or 10, from the mature polypeptide of SEQ ID
NO: 10.
[0178] In another aspect, the parent has a sequence identity to the
mature polypeptide of SEQ ID NO: 12 of at least 60%, e.g., at least
65%, at least 70%, at least 75%, at least 80%, at least 81%, at
least 82%, at least 83%, at least 84%, at least 85%, at least 86%,
at least 87%, at least 88%, at least 89%, at least 90%, at least
91%, at least 92%, at least 93%, at least 94%, at least 95%, at
least 96%, at least 97%, at least 98%, at least 99%, or 100%, which
have cellobiohydrolase activity. In another aspect, the amino acid
sequence of the parent differs by up to 10 amino acids, e.g., 1, 2,
3, 4, 5, 6, 7, 8, 9, or 10, from the mature polypeptide of SEQ ID
NO: 12.
[0179] In another aspect, the parent has a sequence identity to the
mature polypeptide of SEQ ID NO: 14 of at least 60%, e.g., at least
65%, at least 70%, at least 75%, at least 80%, at least 81%, at
least 82%, at least 83%, at least 84%, at least 85%, at least 86%,
at least 87%, at least 88%, at least 89%, at least 90%, at least
91%, at least 92%, at least 93%, at least 94%, at least 95%, at
least 96%, at least 97%, at least 98%, at least 99%, or 100%, which
have cellobiohydrolase activity. In another aspect, the amino acid
sequence of the parent differs by up to 10 amino acids, e.g., 1, 2,
3, 4, 5, 6, 7, 8, 9, or 10, from the mature polypeptide of SEQ ID
NO: 14.
[0180] In another aspect, the parent has a sequence identity to the
mature polypeptide of SEQ ID NO: 16 of at least 60%, e.g., at least
65%, at least 70%, at least 75%, at least 80%, at least 81%, at
least 82%, at least 83%, at least 84%, at least 85%, at least 86%,
at least 87%, at least 88%, at least 89%, at least 90%, at least
91%, at least 92%, at least 93%, at least 94%, at least 95%, at
least 96%, at least 97%, at least 98%, at least 99%, or 100%, which
have cellobiohydrolase activity. In another aspect, the amino acid
sequence of the parent differs by up to 10 amino acids, e.g., 1, 2,
3, 4, 5, 6, 7, 8, 9, or 10, from the mature polypeptide of SEQ ID
NO: 16.
[0181] In another aspect, the parent has a sequence identity to the
mature polypeptide of SEQ ID NO: 18 of at least 60%, e.g., at least
65%, at least 70%, at least 75%, at least 80%, at least 81%, at
least 82%, at least 83%, at least 84%, at least 85%, at least 86%,
at least 87%, at least 88%, at least 89%, at least 90%, at least
91%, at least 92%, at least 93%, at least 94%, at least 95%, at
least 96%, at least 97%, at least 98%, at least 99%, or 100%, which
have cellobiohydrolase activity. In another aspect, the amino acid
sequence of the parent differs by up to 10 amino acids, e.g., 1, 2,
3, 4, 5, 6, 7, 8, 9, or 10, from the mature polypeptide of SEQ ID
NO: 18.
[0182] In another aspect, the parent has a sequence identity to the
mature polypeptide of SEQ ID NO: 20 of at least 60%, e.g., at least
65%, at least 70%, at least 75%, at least 80%, at least 81%, at
least 82%, at least 83%, at least 84%, at least 85%, at least 86%,
at least 87%, at least 88%, at least 89%, at least 90%, at least
91%, at least 92%, at least 93%, at least 94%, at least 95%, at
least 96%, at least 97%, at least 98%, at least 99%, or 100%, which
have cellobiohydrolase activity. In another aspect, the amino acid
sequence of the parent differs by up to 10 amino acids, e.g., 1, 2,
3, 4, 5, 6, 7, 8, 9, or 10, from the mature polypeptide of SEQ ID
NO: 20.
[0183] In another aspect, the parent has a sequence identity to the
mature polypeptide of SEQ ID NO: 22 of at least 60%, e.g., at least
65%, at least 70%, at least 75%, at least 80%, at least 81%, at
least 82%, at least 83%, at least 84%, at least 85%, at least 86%,
at least 87%, at least 88%, at least 89%, at least 90%, at least
91%, at least 92%, at least 93%, at least 94%, at least 95%, at
least 96%, at least 97%, at least 98%, at least 99%, or 100%, which
have cellobiohydrolase activity. In another aspect, the amino acid
sequence of the parent differs by up to 10 amino acids, e.g., 1, 2,
3, 4, 5, 6, 7, 8, 9, or 10, from the mature polypeptide of SEQ ID
NO: 22.
[0184] In another aspect, the parent has a sequence identity to the
mature polypeptide of SEQ ID NO: 52 of at least 60%, e.g., at least
65%, at least 70%, at least 75%, at least 80%, at least 81%, at
least 82%, at least 83%, at least 84%, at least 85%, at least 86%,
at least 87%, at least 88%, at least 89%, at least 90%, at least
91%, at least 92%, at least 93%, at least 94%, at least 95%, at
least 96%, at least 97%, at least 98%, at least 99%, or 100%, which
have cellobiohydrolase activity. In another aspect, the amino acid
sequence of the parent differs by up to 10 amino acids, e.g., 1, 2,
3, 4, 5, 6, 7, 8, 9, or 10, from the mature polypeptide of SEQ ID
NO: 52.
[0185] In another aspect, the parent comprises or consists of the
amino acid sequence of SEQ ID NO: 4. In another aspect, the parent
comprises or consists of the mature polypeptide of SEQ ID NO: 4. In
another aspect, the parent comprises or consists of amino acids 1
to 507 of SEQ ID NO: 4. In another aspect, the parent comprises a
catalytic domain having amino acids 1 to 437 of SEQ ID NO: 4.
[0186] In another aspect, the parent comprises or consists of the
amino acid sequence of SEQ ID NO: 6. In another aspect, the parent
comprises or consists of the mature polypeptide of SEQ ID NO: 6. In
another aspect, the parent comprises or consists of amino acids 1
to 497 of SEQ ID NO: 6. In another aspect, the parent comprises a
catalytic domain having amino acids 1 to 429 of SEQ ID NO: 6.
[0187] In another aspect, the parent comprises or consists of the
amino acid sequence of SEQ ID NO: 8. In another aspect, the parent
comprises or consists of the mature polypeptide of SEQ ID NO: 8. In
another aspect, the parent comprises or consists of amino acids 1
to 440 of SEQ ID NO: 8. In another aspect, the parent comprises a
catalytic domain having amino acids 1 to 440 of SEQ ID NO: 8.
[0188] In another aspect, the parent comprises or consists of the
amino acid sequence of SEQ ID NO: 10. In another aspect, the parent
comprises or consists of the mature polypeptide of SEQ ID NO: 10.
In another aspect, the parent comprises or consists of amino acids
1 to 437 of SEQ ID NO: 10. In another aspect, the parent comprises
a catalytic domain having amino acids 1 to 437 of SEQ ID NO:
10.
[0189] In another aspect, the parent comprises or consists of the
amino acid sequence of SEQ ID NO: 12. In another aspect, the parent
comprises or consists of the mature polypeptide of SEQ ID NO: 12.
In another aspect, the parent comprises or consists of amino acids
1 to 507 of SEQ ID NO: 12. In another aspect, the parent comprises
a catalytic domain having amino acids 1 to 437 of SEQ ID NO:
12.
[0190] In another aspect, the parent comprises or consists of the
amino acid sequence of SEQ ID NO: 14. In another aspect, the parent
comprises or consists of the mature polypeptide of SEQ ID NO: 14.
In another aspect, the parent comprises or consists of amino acids
1 to 507 of SEQ ID NO: 14. In another aspect, the parent comprises
a catalytic domain having amino acids 1 to 438 of SEQ ID NO:
14.
[0191] In another aspect, the parent comprises or consists of the
amino acid sequence of SEQ ID NO: 16. In another aspect, the parent
comprises or consists of the mature polypeptide of SEQ ID NO: 16.
In another aspect, the parent comprises or consists of amino acids
1 to 437 of SEQ ID NO: 16. In another aspect, the parent comprises
a catalytic domain having amino acids 1 to 437 of SEQ ID NO:
16.
[0192] In another aspect, the parent comprises or consists of the
amino acid sequence of SEQ ID NO: 18. In another aspect, the parent
comprises or consists of the mature polypeptide of SEQ ID NO: 18.
In another aspect, the parent comprises or consists of amino acids
1 to 430 of SEQ ID NO: 18. In another aspect, the parent comprises
a catalytic domain having amino acids 1 to 430 of SEQ ID NO:
18.
[0193] In another aspect, the parent comprises or consists of the
amino acid sequence of SEQ ID NO: 20. In another aspect, the parent
comprises or consists of the mature polypeptide of SEQ ID NO: 20.
In another aspect, the parent comprises or consists of amino acids
1 to 511 of SEQ ID NO: 20. In another aspect, the parent comprises
a catalytic domain having amino acids 1 to 433 of SEQ ID NO:
20.
[0194] In another aspect, the parent comprises or consists of the
amino acid sequence of SEQ ID NO: 22. In another aspect, the parent
comprises or consists of the mature polypeptide of SEQ ID NO: 22.
In another aspect, the parent comprises or consists of amino acids
1 to 507 of SEQ ID NO: 22. In another aspect, the parent comprises
a catalytic domain having amino acids 1 to 438 of SEQ ID NO:
22.
[0195] In another aspect, the parent comprises or consists of the
amino acid sequence of SEQ ID NO: 52. In another aspect, the parent
comprises or consists of the mature polypeptide of SEQ ID NO: 52.
In another aspect, the parent comprises or consists of amino acids
1 to 503 of SEQ ID NO: 52. In another aspect, the parent comprises
a catalytic domain having amino acids 1 to 437 of SEQ ID NO:
52.
[0196] In another aspect, the parent is a fragment of the mature
polypeptide of SEQ ID NO: 4 containing at least 430 amino acid
residues, e.g., at least 455 amino acid residues or at least 480
amino acid residues.
[0197] In another aspect, the parent is a fragment of the mature
polypeptide of SEQ ID NO: 6 containing at 420 amino acid residues,
e.g., at least 450 amino acid residues or at least 470 amino acid
residues.
[0198] In another aspect, the parent is a fragment of the mature
polypeptide of SEQ ID NO: 8 containing at 375 amino acid residues,
e.g., at least 400 amino acid residues or at least 420 amino acid
residues.
[0199] In another aspect, the parent is a fragment of the mature
polypeptide of SEQ ID NO: 10 containing at least 370 amino acid
residues, e.g., at least 390 amino acid residues or at least 415
amino acid residues.
[0200] In another aspect, the parent is a fragment of the mature
polypeptide of SEQ ID NO: 12 containing at least 430 amino acid
residues, e.g., at least 455 amino acid residues or at least 480
amino acid residues.
[0201] In another aspect, the parent is a fragment of the mature
polypeptide of SEQ ID NO: 14 containing at least 430 amino acid
residues, e.g., at least 455 amino acid residues or at least 480
amino acid residues.
[0202] In another aspect, the parent is a fragment of the mature
polypeptide of SEQ ID NO: 16 containing at least 370 amino acid
residues, e.g., at least 390 amino acid residues or at least 415
amino acid residues.
[0203] In another aspect, the parent is a fragment of the mature
polypeptide of SEQ ID NO: 18 containing at least 365 amino acid
residues, e.g., at least 390 amino acid residues or at least 410
amino acid residues.
[0204] In another aspect, the parent is a fragment of the mature
polypeptide of SEQ ID NO: 20 containing at least 430 amino acid
residues, e.g., at least 455 amino acid residues or at least 480
amino acid residues.
[0205] In another aspect, the parent is a fragment of the mature
polypeptide of SEQ ID NO: 22 containing at least 430 amino acid
residues, e.g., at least 455 amino acid residues or at least 480
amino acid residues.
[0206] In another aspect, the parent is a fragment of the mature
polypeptide of SEQ ID NO: 52 containing at least 430 amino acid
residues, e.g., at least 455 amino acid residues or at least 480
amino acid residues.
[0207] In another aspect, the parent is encoded by a polynucleotide
that hybridizes under very low stringency conditions, low
stringency conditions, medium stringency conditions, medium-high
stringency conditions, high stringency conditions, or very high
stringency conditions with (i) the mature polypeptide coding
sequence of SEQ ID NO: 1; (ii) the cDNA sequence thereof, or (iii)
the full-length complement of (i) or (ii) (Sambrook et al., 1989,
Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring
Harbor, New York).
[0208] In another aspect, the parent is encoded by a polynucleotide
that hybridizes under low stringency conditions, medium stringency
conditions, medium-high stringency conditions, high stringency
conditions, or very high stringency conditions with (i) the mature
polypeptide coding sequence of SEQ ID NO: 5; (ii) the cDNA sequence
thereof, or (iii) the full-length complement of (i) or (ii)
(Sambrook et al., 1989, supra).
[0209] In another aspect, the parent is encoded by a polynucleotide
that hybridizes under low stringency conditions, medium stringency
conditions, medium-high stringency conditions, high stringency
conditions, or very high stringency conditions with (i) the mature
polypeptide coding sequence of SEQ ID NO: 7; (ii) the cDNA sequence
thereof, or (iii) the full-length complement of (i) or (ii)
(Sambrook et al., 1989, supra).
[0210] In another aspect, the parent is encoded by a polynucleotide
that hybridizes under low stringency conditions, medium stringency
conditions, medium-high stringency conditions, high stringency
conditions, or very high stringency conditions with (i) the mature
polypeptide coding sequence of SEQ ID NO: 9; (ii) the cDNA sequence
thereof, or (iii) the full-length complement of (i) or (ii)
(Sambrook et al., 1989, supra).
[0211] In another aspect, the parent is encoded by a polynucleotide
that hybridizes under low stringency conditions, medium stringency
conditions, medium-high stringency conditions, high stringency
conditions, or very high stringency conditions with (i) the mature
polypeptide coding sequence of SEQ ID NO: 11; (ii) the cDNA
sequence thereof, or (iii) the full-length complement of (i) or
(ii) (Sambrook et al., 1989, supra).
[0212] In another aspect, the parent is encoded by a polynucleotide
that hybridizes under low stringency conditions, medium stringency
conditions, medium-high stringency conditions, high stringency
conditions, or very high stringency conditions with (i) the mature
polypeptide coding sequence of SEQ ID NO: 13; (ii) the cDNA
sequence thereof, or (iii) the full-length complement of (i) or
(ii) (Sambrook et al., 1989, supra).
[0213] In another aspect, the parent is encoded by a polynucleotide
that hybridizes under low stringency conditions, medium stringency
conditions, medium-high stringency conditions, high stringency
conditions, or very high stringency conditions with (i) the mature
polypeptide coding sequence of SEQ ID NO: 15; (ii) the cDNA
sequence thereof, or (iii) the full-length complement of (i) or
(ii) (Sambrook et al., 1989, supra).
[0214] In another aspect, the parent is encoded by a polynucleotide
that hybridizes under low stringency conditions, medium stringency
conditions, medium-high stringency conditions, high stringency
conditions, or very high stringency conditions with (i) the mature
polypeptide coding sequence of SEQ ID NO: 17; (ii) the cDNA
sequence thereof, or (iii) the full-length complement of (i) or
(ii) (Sambrook et al., 1989, supra).
[0215] In another aspect, the parent is encoded by a polynucleotide
that hybridizes under low stringency conditions, medium stringency
conditions, medium-high stringency conditions, high stringency
conditions, or very high stringency conditions with (i) the mature
polypeptide coding sequence of SEQ ID NO: 19; (ii) the cDNA
sequence thereof, or (iii) the full-length complement of (i) or
(ii) (Sambrook et al., 1989, supra).
[0216] In another aspect, the parent is encoded by a polynucleotide
that hybridizes under low stringency conditions, medium stringency
conditions, medium-high stringency conditions, high stringency
conditions, or very high stringency conditions with (i) the mature
polypeptide coding sequence of SEQ ID NO: 21; (ii) the cDNA
sequence thereof, or (iii) the full-length complement of (i) or
(ii) (Sambrook et al., 1989, supra).
[0217] In another aspect, the parent is encoded by a polynucleotide
that hybridizes under low stringency conditions, medium stringency
conditions, medium-high stringency conditions, high stringency
conditions, or very high stringency conditions with (i) the mature
polypeptide coding sequence of SEQ ID NO: 51; (ii) the cDNA
sequence thereof, or (iii) the full-length complement of (i) or
(ii) (Sambrook et al., 1989, supra).
[0218] The polynucleotide of SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO:
7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ
ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 51, or a
subsequence thereof, as well as the polypeptide of SEQ ID NO: 4,
SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID
NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22,
SEQ ID NO: 51 or a fragment thereof, may be used to design nucleic
acid probes to identify and clone DNA encoding a parent from
strains of different genera or species according to methods well
known in the art. In particular, such probes can be used for
hybridization with the genomic DNA or cDNA of a cell of interest,
following standard Southern blotting procedures, in order to
identify and isolate the corresponding gene therein. Such probes
can be considerably shorter than the entire sequence, but should be
at least 15, e.g., at least 25, at least 35, or at least 70
nucleotides in length. Preferably, the nucleic acid probe is at
least 100 nucleotides in length, e.g., at least 200 nucleotides, at
least 300 nucleotides, at least 400 nucleotides, at least 500
nucleotides, at least 600 nucleotides, at least 700 nucleotides, at
least 800 nucleotides, or at least 900 nucleotides in length. Both
DNA and RNA probes can be used. The probes are typically labeled
for detecting the corresponding gene (for example, with .sup.32P,
.sup.3H, .sup.35S, biotin, or avidin). Such probes are encompassed
by the present invention.
[0219] A genomic DNA or cDNA library prepared from such other
strains may be screened for DNA that hybridizes with the probes
described above and encodes a parent. Genomic or other DNA from
such other strains may be separated by agarose or polyacrylamide
gel electrophoresis, or other separation techniques. DNA from the
libraries or the separated DNA may be transferred to and
immobilized on nitrocellulose or other suitable carrier material.
In order to identify a clone or DNA that hybridizes with SEQ ID NO:
1, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID
NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21,
SEQ ID NO: 51, or a subsequence thereof, the carrier material is
used in a Southern blot.
[0220] For purposes of the present invention, hybridization
indicates that the polynucleotide hybridizes to a labeled nucleic
acid probe corresponding to (i) SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID
NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15,
SEQ ID NO: 17, SEQ ID NO: 19, or SEQ ID NO: 21; (ii) the mature
polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID
NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15,
SEQ ID NO: 17, SEQ ID NO: 19, or SEQ ID NO: 21; (iii) the
full-length complement thereof; or (iv) a subsequence thereof;
under very low to very high stringency conditions. Molecules to
which the nucleic acid probe hybridizes under these conditions can
be detected using, for example, X-ray film or any other detection
means known in the art.
[0221] In one aspect, the nucleic acid probe is the mature
polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID
NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15,
SEQ ID NO: 17, SEQ ID NO: 19, or SEQ ID NO: 21. In another aspect,
the nucleic acid probe is nucleotides 79 to 1596 of SEQ ID NO: 1,
nucleotides 52 to 1673 of SEQ ID NO: 5, nucleotides 52 to 1371 of
SEQ ID NO: 7, nucleotides 55 to 1425 of SEQ ID NO: 9, nucleotides
76 to 1596 of SEQ ID NO: 11, nucleotides 76 to 1596 of SEQ ID NO:
13, nucleotides 55 to 1504 of SEQ ID NO: 15, nucleotides 61 to 1350
of SEQ ID NO: 17, nucleotides 55 to 1587 of SEQ ID NO: 19, or
nucleotides 55 to 1575 of SEQ ID NO: 21. In another aspect, the
nucleic acid probe is a polynucleotide that encodes the polypeptide
of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID
NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20,
or SEQ ID NO: 22; the mature polypeptide thereof; or a fragment
thereof. In another aspect, the nucleic acid probe is SEQ ID NO: 1,
SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO:
13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, or SEQ ID NO:
21.
[0222] In another aspect, the parent is encoded by a polynucleotide
having a sequence identity to the mature polypeptide coding
sequence of SEQ ID NO: 1, or the cDNA sequence thereof, of at least
60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%,
at least 81%, at least 82%, at least 83%, at least 84%, at least
85%, at least 86%, at least 87%, at least 88%, at least 89%, at
least 90%, at least 91%, at least 92%, at least 93%, at least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, at least
99%, or 100%.
[0223] In another aspect, the parent is encoded by a polynucleotide
having a sequence identity to the mature polypeptide coding
sequence of SEQ ID NO: 5, or the cDNA sequence thereof, of at least
60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%,
at least 81%, at least 82%, at least 83%, at least 84%, at least
85%, at least 86%, at least 87%, at least 88%, at least 89%, at
least 90%, at least 91%, at least 92%, at least 93%, at least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, at least
99%, or 100%.
[0224] In another aspect, the parent is encoded by a polynucleotide
having a sequence identity to the mature polypeptide coding
sequence of SEQ ID NO: 7, or the cDNA sequence thereof, of at least
60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%,
at least 81%, at least 82%, at least 83%, at least 84%, at least
85%, at least 86%, at least 87%, at least 88%, at least 89%, at
least 90%, at least 91%, at least 92%, at least 93%, at least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, at least
99%, or 100%.
[0225] In another aspect, the parent is encoded by a polynucleotide
having a sequence identity to the mature polypeptide coding
sequence of SEQ ID NO: 9, or the cDNA sequence thereof, of at least
60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%,
at least 81%, at least 82%, at least 83%, at least 84%, at least
85%, at least 86%, at least 87%, at least 88%, at least 89%, at
least 90%, at least 91%, at least 92%, at least 93%, at least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, at least
99%, or 100%.
[0226] In another aspect, the parent is encoded by a polynucleotide
having a sequence identity to the mature polypeptide coding
sequence of SEQ ID NO: 11, or the cDNA sequence thereof, of at
least 60%, e.g., at least 65%, at least 70%, at least 75%, at least
80%, at least 81%, at least 82%, at least 83%, at least 84%, at
least 85%, at least 86%, at least 87%, at least 88%, at least 89%,
at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99%, or 100%.
[0227] In another aspect, the parent is encoded by a polynucleotide
having a sequence identity to the mature polypeptide coding
sequence of SEQ ID NO: 13, or the cDNA sequence thereof, of at
least 60%, e.g., at least 65%, at least 70%, at least 75%, at least
80%, at least 81%, at least 82%, at least 83%, at least 84%, at
least 85%, at least 86%, at least 87%, at least 88%, at least 89%,
at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99%, or 100%.
[0228] In another aspect, the parent is encoded by a polynucleotide
having a sequence identity to the mature polypeptide coding
sequence of SEQ ID NO: 15, or the cDNA sequence thereof, of at
least 60%, e.g., at least 65%, at least 70%, at least 75%, at least
80%, at least 81%, at least 82%, at least 83%, at least 84%, at
least 85%, at least 86%, at least 87%, at least 88%, at least 89%,
at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99%, or 100%.
[0229] In another aspect, the parent is encoded by a polynucleotide
having a sequence identity to the mature polypeptide coding
sequence of SEQ ID NO: 17, or the cDNA sequence thereof, of at
least 60%, e.g., at least 65%, at least 70%, at least 75%, at least
80%, at least 81%, at least 82%, at least 83%, at least 84%, at
least 85%, at least 86%, at least 87%, at least 88%, at least 89%,
at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99%, or 100%.
[0230] In another aspect, the parent is encoded by a polynucleotide
having a sequence identity to the mature polypeptide coding
sequence of SEQ ID NO: 19, or the cDNA sequence thereof, of at
least 60%, e.g., at least 65%, at least 70%, at least 75%, at least
80%, at least 81%, at least 82%, at least 83%, at least 84%, at
least 85%, at least 86%, at least 87%, at least 88%, at least 89%,
at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99%, or 100%.
[0231] In another aspect, the parent is encoded by a polynucleotide
having a sequence identity to the mature polypeptide coding
sequence of SEQ ID NO: 21, or the cDNA sequence thereof, of at
least 60%, e.g., at least 65%, at least 70%, at least 75%, at least
80%, at least 81%, at least 82%, at least 83%, at least 84%, at
least 85%, at least 86%, at least 87%, at least 88%, at least 89%,
at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99%, or 100%.
[0232] In another embodiment, the parent is an allelic variant of
the mature polypeptide of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8,
SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID
NO: 18, SEQ ID NO: 20, or SEQ ID NO: 22.
[0233] The parent may also be a hybrid or chimeric polypeptide in
which a region of parent is replaced with a region of another
polypeptide. In one aspect, the region is a carbohydrate binding
domain. The carbohydrate binding domain of a parent may be replaced
with another (heterologous) carbohydrate binding domain, as
discussed supra.
[0234] The parent may also be a fusion polypeptide or cleavable
fusion polypeptide in which another polypeptide is fused at the
N-terminus or the C-terminus of the parent. In one aspect, the
other polypeptide is a carbohydrate binding domain. The catalytic
domain of a parent without a carbohydrate binding domain may be
fused to a carbohydrate binding domain. A fusion polypeptide is
produced by fusing a polynucleotide encoding another polypeptide to
a polynucleotide encoding a parent. Techniques for producing fusion
polypeptides are described supra. A fusion polypeptide can further
comprise a cleavage site between the two polypeptides as described
supra.
[0235] In one embodiment, the parent is a hybrid polypeptide in
which the carbohydrate binding domain of the parent is replaced
with a different carbohydrate binding domain. In another
embodiment, the parent is a fusion protein in which a heterologous
carbohydrate binding domain is fused to the parent without a
carbohydrate binding domain. For example, the parent may comprise a
Rasamsonia emersonii cellobiohydrolase catalytic domain fused to
the Trichoderma reesei carbohydrate binding domain. Accordingly, in
one aspect, the parent has a sequence identity to the mature
polypeptide of SEQ ID NO: 24 of at least 60%, e.g., at least 65%,
at least 70%, at least 75%, at least 80%, at least 81%, at least
82%, at least 83%, at least 84%, at least 85%, at least 86%, at
least 87%, at least 88%, at least 89%, at least 90%, at least 91%,
at least 92%, at least 93%, at least 94%, at least 95%, at least
96%, at least 97%, at least 98%, at least 99%, or 100%, which have
cellobiohydrolase activity. In another aspect, the amino acid
sequence of the parent differs by up to 10 amino acids, e.g., 1, 2,
3, 4, 5, 6, 7, 8, 9, or 10, from the mature polypeptide of SEQ ID
NO: 24. In one aspect, the carbohydrate binding domain is fused to
the N-terminus of the variant. In another aspect, the carbohydrate
binding domain is fused to the C-terminus of the variant.
[0236] The parent may be obtained from microorganisms of any genus.
For purposes of the present invention, the term "obtained from" as
used herein in connection with a given source shall mean that the
parent encoded by a polynucleotide is produced by the source or by
a strain in which the polynucleotide from the source has been
inserted. In one aspect, the parent is secreted
extracellularly.
[0237] The parent may be a filamentous fungal cellobiohydrolase.
For example, the parent may be a filamentous fungal
cellobiohydrolase such as an Aspergillus, Chaetomium,
Chrysosporium, Myceliophthora, Penicillium, Talaromyces,
Thermoascus, or Trichoderma cellobiohydrolase.
[0238] In one aspect, the parent is an Aspergillus aculeatus,
Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus,
Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger,
Aspergillus oryzae, Chaetomium thermophilum, Chrysosporium inops,
Chrysosporium keratinophilum, Chrysosporium lucknowense,
Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium
queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum,
Myceliophthora thermophila, Penicillium emersonii, Penicillium
funiculosum, Penicillium purpurogenum, Talaromyces
byssochlamydoides, Talaromyces emersonii, Talaromyces leycettanus,
Trichoderma harzianum, Trichoderma koningii, Trichoderma
longibrachiatum, Trichoderma reesei, or Trichoderma viride
cellobiohydrolase.
[0239] In another aspect, the parent is an Aspergillus fumigatus
cellobiohydrolase, e.g., the cellobiohydrolase of SEQ ID NO: 4 or
the mature polypeptide thereof.
[0240] In another aspect, the parent is a Trichoderma reesei
cellobiohydrolase, e.g., the cellobiohydrolase of SEQ ID NO: 6 or
the mature polypeptide thereof.
[0241] In another aspect, the parent is a Thermoascus aurantiacus
cellobiohydrolase, e.g., the cellobiohydrolase of SEQ ID NO: 8 or
the mature polypeptide thereof.
[0242] In another aspect, the parent is a Penicillium emersonii
(Rasamsonia emersonii) cellobiohydrolase, e.g., the
cellobiohydrolase of SEQ ID NO: 10 or the mature polypeptide
thereof.
[0243] In another aspect, the parent is a Talaromyces leycettanus
cellobiohydrolase, e.g., the cellobiohydrolase of SEQ ID NO: 12,
SEQ ID NO: 14, or the mature polypeptide thereof.
[0244] In another aspect, the parent is a Talaromyces
byssochlamydoides cellobiohydrolase, e.g., the cellobiohydrolase of
SEQ ID NO: 16 or the mature polypeptide thereof.
[0245] In another aspect, the parent is a Myceliophthora
thermophila cellobiohydrolase, e.g., the cellobiohydrolase of SEQ
ID NO: 18 or the mature polypeptide thereof.
[0246] In another aspect, the parent is a Chaetomium thermophilum
cellobiohydrolase, e.g., the cellobiohydrolase of SEQ ID NO: 20 or
the mature polypeptide thereof.
[0247] In another aspect, the parent is a Humicola grisea
cellobiohydrolase, e.g., the cellobiohydrolase of SEQ ID NO: 22 or
the mature polypeptide thereof.
[0248] It will be understood that for the aforementioned species,
the invention encompasses both the perfect and imperfect states,
and other taxonomic equivalents, e.g., anamorphs, regardless of the
species name by which they are known. Those skilled in the art will
readily recognize the identity of appropriate equivalents.
[0249] Strains of these species are readily accessible to the
public in a number of culture collections, such as the American
Type Culture Collection (ATCC), Deutsche Sammlung von
Mikroorganismen and Zellkulturen GmbH (DSMZ), Centraalbureau Voor
Schimmelcultures (CBS), and Agricultural Research Service Patent
Culture Collection, Northern Regional Research Center (NRRL).
[0250] The parent may be identified and obtained from other sources
including microorganisms isolated from nature (e.g., soil,
composts, water, etc.) or DNA samples obtained directly from
natural materials (e.g., soil, composts, water, etc.) using the
above-mentioned probes. Techniques for isolating microorganisms and
DNA directly from natural habitats are well known in the art. A
polynucleotide encoding a parent may then be obtained by similarly
screening a genomic DNA or cDNA library of another microorganism or
mixed DNA sample. Once a polynucleotide encoding a parent has been
detected with the probe(s), the polynucleotide can be isolated or
cloned by utilizing techniques that are known to those of ordinary
skill in the art (see, e.g., Sambrook et al., 1989, supra).
Preparation of Variants
[0251] The present invention also relates to methods for obtaining
a cellobiohydrolase variant, comprising: (a) introducing into a
parent cellobiohydrolase an alteration at one or more (e.g.,
several) positions corresponding to positions 4, 44, 45, 72, 265,
266, 391, 393 and 394 of SEQ ID NO: 4, wherein the variant has
cellobiohydrolase activity; and optionally (b) recovering the
variant.
[0252] The variants can be prepared using any mutagenesis procedure
known in the art, such as site-directed mutagenesis, synthetic gene
construction, semi-synthetic gene construction, random mutagenesis,
shuffling, etc.
[0253] Site-directed mutagenesis is a technique in which one or
more (e.g., several) mutations are introduced at one or more
defined sites in a polynucleotide encoding the parent. Any
site-directed mutagenesis procedure can be used in the present
invention.
[0254] There are many commercial kits available that can be used to
prepare variants. Site-directed mutagenesis can be accomplished in
vitro by PCR involving the use of oligonucleotide primers
containing the desired mutation. Site-directed mutagenesis can also
be performed in vitro by cassette mutagenesis involving the
cleavage by a restriction enzyme at a site in the plasmid
comprising a polynucleotide encoding the parent and subsequent
ligation of an oligonucleotide containing the mutation in the
polynucleotide. Usually the restriction enzyme that digests the
plasmid and the oligonucleotide is the same, permitting sticky ends
of the plasmid and the insert to ligate to one another. See, e.g.,
Scherer and Davis, 1979, Proc. Natl. Acad. Sci. USA 76: 4949-4955;
and Barton et al., 1990, Nucleic Acids Res. 18: 7349-4966.
[0255] Site-directed mutagenesis can also be accomplished in vivo
by methods known in the art. See, e.g., U.S. Patent Application
Publication No. 2004/0171154; Storici et al., 2001, Nature
Biotechnol. 19: 773-776; Kren et al., 1998, Nat. Med. 4: 285-290;
and Calissano and Macino, 1996, Fungal Genet. Newslett. 43:
15-16.
[0256] Site-saturation mutagenesis systematically replaces a
polypeptide coding sequence with sequences encoding all 19 amino
acids at one or more (e.g., several) specific positions (Parikh and
Matsumura, 2005, J. Mol. Biol. 352: 621-628).
[0257] Synthetic gene construction entails in vitro synthesis of a
designed polynucleotide molecule to encode a polypeptide of
interest. Gene synthesis can be performed utilizing a number of
techniques, such as the multiplex microchip-based technology
described by Tian et al. (2004, Nature 432: 1050-1054) and similar
technologies wherein oligonucleotides are synthesized and assembled
upon photo-programmable microfluidic chips.
[0258] Single or multiple amino acid substitutions, deletions,
and/or insertions can be made and tested using known methods of
mutagenesis, recombination, and/or shuffling, followed by a
relevant screening procedure, such as those disclosed by
Reidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and
Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413;
or WO 95/22625. Other methods that can be used include error-prone
PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30:
10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204) and
region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145;
Ner et al., 1988, DNA 7: 127).
[0259] Mutagenesis/shuffling methods can be combined with
high-throughput, automated screening methods to detect activity of
cloned, mutagenized polypeptides expressed by host cells (Ness et
al., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA
molecules that encode active polypeptides can be recovered from the
host cells and rapidly sequenced using standard methods in the art.
These methods allow the rapid determination of the importance of
individual amino acid residues in a polypeptide.
[0260] Semi-synthetic gene construction is accomplished by
combining aspects of synthetic gene construction, and/or
site-directed mutagenesis, and/or random mutagenesis, and/or
shuffling. Semi-synthetic construction is typified by a process
utilizing polynucleotide fragments that are synthesized, in
combination with PCR techniques. Defined regions of genes may thus
be synthesized de novo, while other regions may be amplified using
site-specific mutagenic primers, while yet other regions may be
subjected to error-prone PCR or non-error prone PCR amplification.
Polynucleotide subsequences may then be shuffled.
Polynucleotides
[0261] The present invention also relates to isolated
polynucleotides encoding a variant of the present invention.
Nucleic Acid Constructs
[0262] The present invention also relates to nucleic acid
constructs comprising a polynucleotide encoding a variant of the
present invention operably linked to one or more control sequences
that direct the expression of the coding sequence in a suitable
host cell under conditions compatible with the control
sequences.
[0263] The polynucleotide may be manipulated in a variety of ways
to provide for expression of a variant. Manipulation of the
polynucleotide prior to its insertion into a vector may be
desirable or necessary depending on the expression vector. The
techniques for modifying polynucleotides utilizing recombinant DNA
methods are well known in the art.
[0264] The control sequence may be a promoter, a polynucleotide
recognized by a host cell for expression of a polynucleotide
encoding a variant of the present invention. The promoter contains
transcriptional control sequences that mediate the expression of
the variant. The promoter may be any polynucleotide that shows
transcriptional activity in the host cell including mutant,
truncated, and hybrid promoters, and may be obtained from genes
encoding extracellular or intracellular polypeptides either
homologous or heterologous to the host cell.
[0265] Examples of suitable promoters for directing transcription
of the nucleic acid constructs of the present invention in a
bacterial host cell are the promoters obtained from the Bacillus
amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis
alpha-amylase gene (amyL), Bacillus licheniformis penicillinase
gene (penP), Bacillus stearothermophilus maltogenic amylase gene
(amyM), Bacillus subtilis levansucrase gene (sacB), Bacillus
subtilis xylA and xylB genes, Bacillus thuringiensis cryIIIA gene
(Agaisse and Lereclus, 1994, Molecular Microbiology 13: 97-107), E.
coli lac operon, E. coli trc promoter (Egon et al., 1988, Gene 69:
301-315), Streptomyces coelicolor agarase gene (dagA), and
prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978, Proc.
Natl. Acad. Sci. USA 75: 3727-3731), as well as the tac promoter
(DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80: 21-25).
Further promoters are described in "Useful proteins from
recombinant bacteria" in Gilbert et al., 1980, Scientific American
242: 74-94; and in Sambrook et al., 1989, supra. Examples of tandem
promoters are disclosed in WO 99/43835.
[0266] Examples of suitable promoters for directing transcription
of the nucleic acid constructs of the present invention in a
filamentous fungal host cell are promoters obtained from the genes
for Aspergillus nidulans acetamidase, Aspergillus niger neutral
alpha-amylase, Aspergillus niger acid stable alpha-amylase,
Aspergillus niger or Aspergillus awamori glucoamylase (glaA),
Aspergillus oryzae TAKA amylase, Aspergillus oryzae alkaline
protease, Aspergillus oryzae triose phosphate isomerase, Fusarium
oxysporum trypsin-like protease (WO 96/00787), Fusarium venenatum
amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO
00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor
miehei lipase, Rhizomucor miehei aspartic proteinase, Trichoderma
reesei beta-glucosidase, Trichoderma reesei cellobiohydrolase I,
Trichoderma reesei cellobiohydrolase II, Trichoderma reesei
endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma
reesei endoglucanase III, Trichoderma reesei endoglucanase V,
Trichoderma reesei xylanase I, Trichoderma reesei xylanase II,
Trichoderma reesei xylanase III, Trichoderma reesei
beta-xylosidase, and Trichoderma reesei translation elongation
factor, as well as the NA2-tpi promoter (a modified promoter from
an Aspergillus neutral alpha-amylase gene in which the untranslated
leader has been replaced by an untranslated leader from an
Aspergillus triose phosphate isomerase gene; non-limiting examples
include modified promoters from an Aspergillus niger neutral
alpha-amylase gene in which the untranslated leader has been
replaced by an untranslated leader from an Aspergillus nidulans or
Aspergillus oryzae triose phosphate isomerase gene); and mutant,
truncated, and hybrid promoters thereof. Other promoters are
described in U.S. Pat. No. 6,011,147.
[0267] In a yeast host, useful promoters are obtained from the
genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces
cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol
dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1,
ADH2/GAP), Saccharomyces cerevisiae triose phosphate isomerase
(TPI), Saccharomyces cerevisiae metallothionein (CUP1), and
Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful
promoters for yeast host cells are described by Romanos et al.,
1992, Yeast 8: 423-488.
[0268] The control sequence may also be a transcription terminator,
which is recognized by a host cell to terminate transcription. The
terminator is operably linked to the 3'-terminus of the
polynucleotide encoding the variant. Any terminator that is
functional in the host cell may be used in the present
invention.
[0269] Preferred terminators for bacterial host cells are obtained
from the genes for Bacillus clausii alkaline protease (aprH),
Bacillus licheniformis alpha-amylase (amyL), and Escherichia coli
ribosomal RNA (rrnB).
[0270] Preferred terminators for filamentous fungal host cells are
obtained from the genes for Aspergillus nidulans acetamidase,
Aspergillus nidulans anthranilate synthase, Aspergillus niger
glucoamylase, Aspergillus niger alpha-glucosidase, Aspergillus
oryzae TAKA amylase, Fusarium oxysporum trypsin-like protease,
Trichoderma reesei beta-glucosidase, Trichoderma reesei
cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II,
Trichoderma reesei endoglucanase I, Trichoderma reesei
endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma
reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma
reesei xylanase II, Trichoderma reesei xylanase III, Trichoderma
reesei beta-xylosidase, and Trichoderma reesei translation
elongation factor.
[0271] Preferred terminators for yeast host cells are obtained from
the genes for Saccharomyces cerevisiae enolase, Saccharomyces
cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae
glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators
for yeast host cells are described by Romanos et al., 1992,
supra.
[0272] The control sequence may also be an mRNA stabilizer region
downstream of a promoter and upstream of the coding sequence of a
gene which increases expression of the gene.
[0273] Examples of suitable mRNA stabilizer regions are obtained
from a Bacillus thuringiensis cryIIIA gene (WO 94/25612) and a
Bacillus subtilis SP82 gene (Hue et al., 1995, Journal of
Bacteriology 177: 3465-3471).
[0274] The control sequence may also be a leader, a nontranslated
region of an mRNA that is important for translation by the host
cell. The leader is operably linked to the 5'-terminus of the
polynucleotide encoding the variant. Any leader that is functional
in the host cell may be used.
[0275] Preferred leaders for filamentous fungal host cells are
obtained from the genes for Aspergillus oryzae TAKA amylase and
Aspergillus nidulans triose phosphate isomerase.
[0276] Suitable leaders for yeast host cells are obtained from the
genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces
cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae
alpha-factor, and Saccharomyces cerevisiae alcohol
dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase
(ADH2/GAP).
[0277] The control sequence may also be a polyadenylation sequence,
a sequence operably linked to the 3'-terminus of the polynucleotide
and, when transcribed, is recognized by the host cell as a signal
to add polyadenosine residues to transcribed mRNA. Any
polyadenylation sequence that is functional in the host cell may be
used.
[0278] Preferred polyadenylation sequences for filamentous fungal
host cells are obtained from the genes for Aspergillus nidulans
anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus
niger alpha-glucosidase Aspergillus oryzae TAKA amylase, and
Fusarium oxysporum trypsin-like protease.
[0279] Useful polyadenylation sequences for yeast host cells are
described by Guo and Sherman, 1995, Mol. Cellular Biol. 15:
5983-5990.
[0280] The control sequence may also be a signal peptide coding
region that encodes a signal peptide linked to the N-terminus of a
variant and directs the variant into the cell's secretory pathway.
The 5'-end of the coding sequence of the polynucleotide may
inherently contain a signal peptide coding sequence naturally
linked in translation reading frame with the segment of the coding
sequence that encodes the variant. Alternatively, the 5'-end of the
coding sequence may contain a signal peptide coding sequence that
is foreign to the coding sequence. A foreign signal peptide coding
sequence may be required where the coding sequence does not
naturally contain a signal peptide coding sequence. Alternatively,
a foreign signal peptide coding sequence may simply replace the
natural signal peptide coding sequence in order to enhance
secretion of the variant. However, any signal peptide coding
sequence that directs the expressed variant into the secretory
pathway of a host cell may be used.
[0281] Effective signal peptide coding sequences for bacterial host
cells are the signal peptide coding sequences obtained from the
genes for Bacillus NCIB 11837 maltogenic amylase, Bacillus
licheniformis subtilisin, Bacillus licheniformis beta-lactamase,
Bacillus stearothermophilus alpha-amylase, Bacillus
stearothermophilus neutral proteases (nprT, nprS, nprM), and
Bacillus subtilis prsA. Further signal peptides are described by
Simonen and Palva, 1993, Microbiological Reviews 57: 109-137.
[0282] Effective signal peptide coding sequences for filamentous
fungal host cells are the signal peptide coding sequences obtained
from the genes for Aspergillus niger neutral amylase, Aspergillus
niger glucoamylase, Aspergillus oryzae TAKA amylase, Humicola
insolens cellulase, Humicola insolens endoglucanase V, Humicola
lanuginosa lipase, and Rhizomucor miehei aspartic proteinase.
[0283] Useful signal peptides for yeast host cells are obtained
from the genes for Saccharomyces cerevisiae alpha-factor and
Saccharomyces cerevisiae invertase. Other useful signal peptide
coding sequences are described by Romanos et al., 1992, supra.
[0284] The control sequence may also be a propeptide coding
sequence that encodes a propeptide positioned at the N-terminus of
a variant. The resultant polypeptide is known as a proenzyme or
propolypeptide (or a zymogen in some cases). A propolypeptide is
generally inactive and can be converted to an active variant by
catalytic or autocatalytic cleavage of the propeptide from the
propolypeptide. The propeptide coding sequence may be obtained from
the genes for Bacillus subtilis alkaline protease (aprE), Bacillus
subtilis neutral protease (nprT), Myceliophthora thermophila
laccase (WO 95/33836), Rhizomucor miehei aspartic proteinase, and
Saccharomyces cerevisiae alpha-factor.
[0285] Where both signal peptide and propeptide sequences are
present, the propeptide sequence is positioned next to the
N-terminus of a variant and the signal peptide sequence is
positioned next to the N-terminus of the propeptide sequence.
[0286] It may also be desirable to add regulatory sequences that
regulate expression of the variant relative to the growth of the
host cell. Examples of regulatory sequences are those that cause
expression of the gene to be turned on or off in response to a
chemical or physical stimulus, including the presence of a
regulatory compound. Regulatory sequences in prokaryotic systems
include the lac, tac, and trp operator systems. In yeast, the ADH2
system or GAL1 system may be used. In filamentous fungi, the
Aspergillus niger glucoamylase promoter, Aspergillus oryzae TAKA
alpha-amylase promoter, and Aspergillus oryzae glucoamylase
promoter, Trichoderma reesei cellobiohydrolase I promoter, and
Trichoderma reesei cellobiohydrolase II promoter may be used. Other
examples of regulatory sequences are those that allow for gene
amplification. In eukaryotic systems, these regulatory sequences
include the dihydrofolate reductase gene that is amplified in the
presence of methotrexate, and the metallothionein genes that are
amplified with heavy metals. In these cases, the polynucleotide
encoding the variant would be operably linked to the regulatory
sequence.
Expression Vectors
[0287] The present invention also relates to recombinant expression
vectors comprising a polynucleotide encoding a variant of the
present invention, a promoter, and transcriptional and
translational stop signals. The various nucleotide and control
sequences may be joined together to produce a recombinant
expression vector that may include one or more convenient
restriction sites to allow for insertion or substitution of the
polynucleotide encoding the variant at such sites. Alternatively,
the polynucleotide may be expressed by inserting the polynucleotide
or a nucleic acid construct comprising the polynucleotide into an
appropriate vector for expression. In creating the expression
vector, the coding sequence is located in the vector so that the
coding sequence is operably linked with the appropriate control
sequences for expression.
[0288] The recombinant expression vector may be any vector (e.g., a
plasmid or virus) that can be conveniently subjected to recombinant
DNA procedures and can bring about expression of the
polynucleotide. The choice of the vector will typically depend on
the compatibility of the vector with the host cell into which the
vector is to be introduced. The vector may be a linear or closed
circular plasmid.
[0289] The vector may be an autonomously replicating vector, i.e.,
a vector that exists as an extrachromosomal entity, the replication
of which is independent of chromosomal replication, e.g., a
plasmid, an extrachromosomal element, a minichromosome, or an
artificial chromosome. The vector may contain any means for
assuring self-replication. Alternatively, the vector may be one
that, when introduced into the host cell, is integrated into the
genome and replicated together with the chromosome(s) into which it
has been integrated. Furthermore, a single vector or plasmid or two
or more vectors or plasmids that together contain the total DNA to
be introduced into the genome of the host cell, or a transposon,
may be used.
[0290] The vector preferably contains one or more selectable
markers that permit easy selection of transformed, transfected,
transduced, or the like cells. A selectable marker is a gene the
product of which provides for biocide or viral resistance,
resistance to heavy metals, prototrophy to auxotrophs, and the
like.
[0291] Examples of bacterial selectable markers are Bacillus
licheniformis or Bacillus subtilis dal genes, or markers that
confer antibiotic resistance such as ampicillin, chloramphenicol,
kanamycin, neomycin, spectinomycin, or tetracycline resistance.
Suitable markers for yeast host cells include, but are not limited
to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable
markers for use in a filamentous fungal host cell include, but are
not limited to, adeA (phosphoribosylam
inoimidazole-succinocarboxamide synthase), adeB
(phosphoribosyl-aminoimidazole synthase), amdS (acetamidase), argB
(ornithine carbamoyltransferase), bar (phosphinothricin
acetyltransferase), hph (hygromycin phosphotransferase), niaD
(nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase),
sC (sulfate adenyltransferase), and trpC (anthranilate synthase),
as well as equivalents thereof. Preferred for use in an Aspergillus
cell are Aspergillus nidulans or Aspergillus oryzae amdS and pyrG
genes and a Streptomyces hygroscopicus bar gene. Preferred for use
in a Trichoderma cell are adeA, adeB, amdS, hph, and pyrG
genes.
[0292] The selectable marker may be a dual selectable marker system
as described in WO 2010/039889. In one aspect, the dual selectable
marker is a hph-tk dual selectable marker system.
[0293] The vector preferably contains an element(s) that permits
integration of the vector into the host cell's genome or autonomous
replication of the vector in the cell independent of the
genome.
[0294] For integration into the host cell genome, the vector may
rely on the polynucleotide's sequence encoding the variant or any
other element of the vector for integration into the genome by
homologous or non-homologous recombination. Alternatively, the
vector may contain additional polynucleotides for directing
integration by homologous recombination into the genome of the host
cell at a precise location(s) in the chromosome(s). To increase the
likelihood of integration at a precise location, the integrational
elements should contain a sufficient number of nucleic acids, such
as 100 to 10,000 base pairs, 400 to 10,000 base pairs, and 800 to
10,000 base pairs, which have a high degree of sequence identity to
the corresponding target sequence to enhance the probability of
homologous recombination.
[0295] The integrational elements may be any sequence that is
homologous with the target sequence in the genome of the host cell.
Furthermore, the integrational elements may be non-encoding or
encoding polynucleotides. On the other hand, the vector may be
integrated into the genome of the host cell by non-homologous
recombination.
[0296] For autonomous replication, the vector may further comprise
an origin of replication enabling the vector to replicate
autonomously in the host cell in question. The origin of
replication may be any plasmid replicator mediating autonomous
replication that functions in a cell. The term "origin of
replication" or "plasmid replicator" means a polynucleotide that
enables a plasmid or vector to replicate in vivo.
[0297] Examples of bacterial origins of replication are the origins
of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184
permitting replication in E. coli, and pUB110, pE194, pTA1060, and
pAM.beta.1 permitting replication in Bacillus.
[0298] Examples of origins of replication for use in a yeast host
cell are the 2 micron origin of replication, ARS1, ARS4, the
combination of ARS1 and CEN3, and the combination of ARS4 and
CEN6.
[0299] Examples of origins of replication useful in a filamentous
fungal cell are AMA1 and ANSI (Gems et al., 1991, Gene 98: 61-67;
Cullen et al., 1987, Nucleic Acids Res. 15: 9163-9175; WO
00/24883). Isolation of the AMA1 gene and construction of plasmids
or vectors comprising the gene can be accomplished according to the
methods disclosed in WO 00/24883.
[0300] More than one copy of a polynucleotide of the present
invention may be inserted into a host cell to increase production
of a variant. An increase in the copy number of the polynucleotide
can be obtained by integrating at least one additional copy of the
sequence into the host cell genome or by including an amplifiable
selectable marker gene with the polynucleotide where cells
containing amplified copies of the selectable marker gene, and
thereby additional copies of the polynucleotide, can be selected
for by cultivating the cells in the presence of the appropriate
selectable agent.
[0301] The procedures used to ligate the elements described above
to construct the recombinant expression vectors of the present
invention are well known to one skilled in the art (see, e.g.,
Sambrook et al., 1989, supra).
Host Cells
[0302] The present invention also relates to recombinant host
cells, comprising a polynucleotide encoding a variant of the
present invention operably linked to one or more control sequences
that direct the production of a variant of the present invention. A
construct or vector comprising a polynucleotide is introduced into
a host cell so that the construct or vector is maintained as a
chromosomal integrant or as a self-replicating extra-chromosomal
vector as described earlier. The term "host cell" encompasses any
progeny of a parent cell that is not identical to the parent cell
due to mutations that occur during replication. The choice of a
host cell will to a large extent depend upon the gene encoding the
variant and its source.
[0303] The host cell may be any cell useful in the recombinant
production of a variant, e.g., a prokaryote or a eukaryote.
[0304] The prokaryotic host cell may be any Gram-positive or
Gram-negative bacterium. Gram-positive bacteria include, but are
not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus,
Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus,
Streptococcus, and Streptomyces. Gram-negative bacteria include,
but are not limited to, Campylobacter, E. coli, Flavobacterium,
Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas,
Salmonella, and Ureaplasma.
[0305] The bacterial host cell may be any Bacillus cell including,
but not limited to, Bacillus alkalophilus, Bacillus
amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus
clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus,
Bacillus lentus, Bacillus licheniformis, Bacillus megaterium,
Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis,
and Bacillus thuringiensis cells.
[0306] The bacterial host cell may also be any Streptococcus cell
including, but not limited to, Streptococcus equisimilis,
Streptococcus pyogenes, Streptococcus uberis, and Streptococcus
equi subsp. Zooepidemicus cells.
[0307] The bacterial host cell may also be any Streptomyces cell,
including, but not limited to, Streptomyces achromogenes,
Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces
griseus, and Streptomyces lividans cells.
[0308] The introduction of DNA into a Bacillus cell may be effected
by protoplast transformation (see, e.g., Chang and Cohen, 1979,
Mol. Gen. Genet. 168: 111-115), competent cell transformation (see,
e.g., Young and Spizizen, 1961, J. Bacteriol. 81: 823-829, or
Dubnau and Davidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221),
electroporation (see, e.g., Shigekawa and Dower, 1988,
Biotechniques 6: 742-751), or conjugation (see, e.g., Koehler and
Thorne, 1987, J. Bacteriol. 169: 5271-5278). The introduction of
DNA into an E. coli cell may be effected by protoplast
transformation (see, e.g., Hanahan, 1983, J. Mol. Biol. 166:
557-580) or electroporation (see, e.g., Dower et al., 1988, Nucleic
Acids Res. 16: 6127-6145). The introduction of DNA into a
Streptomyces cell may be effected by protoplast transformation,
electroporation (see, e.g., Gong et al., 2004, Folia Microbiol.
(Praha) 49: 399-405), conjugation (see, e.g., Mazodier et al.,
1989, J. Bacteriol. 171: 3583-3585), or transduction (see, e.g.,
Burke et al., 2001, Proc. Natl. Acad. Sci. USA 98: 6289-6294). The
introduction of DNA into a Pseudomonas cell may be effected by
electroporation (see, e.g., Choi et al., 2006, J. Microbiol.
Methods 64: 391-397), or conjugation (see, e.g., Pinedo and Smets,
2005, Appl. Environ. Microbiol. 71: 51-57). The introduction of DNA
into a Streptococcus cell may be effected by natural competence
(see, e.g., Perry and Kuramitsu, 1981, Infect Immun. 32:
1295-1297), protoplast transformation (see, e.g., Catt and Jollick,
1991, Microbios 68: 189-207), electroporation (see, e.g., Buckley
et al., 1999, Appl. Environ. Microbiol. 65: 3800-3804), or
conjugation (see, e.g., Clewell, 1981, Microbiol. Rev. 45:
409-436). However, any method known in the art for introducing DNA
into a host cell can be used.
[0309] The host cell may also be a eukaryote, such as a mammalian,
insect, plant, or fungal cell.
[0310] The host cell may be a fungal cell. "Fungi" as used herein
includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and
Zygomycota as well as the Oomycota and all mitosporic fungi (as
defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary
of The Fungi, 8th edition, 1995, CAB International, University
Press, Cambridge, UK).
[0311] The fungal host cell may be a yeast cell. "Yeast" as used
herein includes ascosporogenous yeast (Endomycetales),
basidiosporogenous yeast, and yeast belonging to the Fungi
Imperfecti (Blastomycetes). Since the classification of yeast may
change in the future, for the purposes of this invention, yeast
shall be defined as described in Biology and Activities of Yeast
(Skinner, Passmore, and Davenport, editors, Soc. App. Bacteriol.
Symposium Series No. 9, 1980).
[0312] The yeast host cell may be a Candida, Hansenula,
Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or
Yarrowia cell such as a Kluyveromyces lactis, Saccharomyces
carlsbergensis, Saccharomyces cerevisiae, Saccharomyces
diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri,
Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia
lipolytica cell.
[0313] The fungal host cell may be a filamentous fungal cell.
"Filamentous fungi" include all filamentous forms of the
subdivision Eumycota and Oomycota (as defined by Hawksworth et al.,
1995, supra). The filamentous fungi are generally characterized by
a mycelial wall composed of chitin, cellulose, glucan, chitosan,
mannan, and other complex polysaccharides. Vegetative growth is by
hyphal elongation and carbon catabolism is obligately aerobic. In
contrast, vegetative growth by yeasts such as Saccharomyces
cerevisiae is by budding of a unicellular thallus and carbon
catabolism may be fermentative.
[0314] The filamentous fungal host cell may be an Acremonium,
Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis,
Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium,
Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora,
Neocallimastix, Neurospora, Paecilomyces, Penicillium,
Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum,
Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or
Trichoderma cell.
[0315] For example, the filamentous fungal host cell may be an
Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus,
Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger,
Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina,
Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis
pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa,
Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium
keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium,
Chrysosporium pannicola, Chrysosporium queenslandicum,
Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus,
Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis,
Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum,
Fusarium graminum, Fusarium heterosporum, Fusarium negundi,
Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium
sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides,
Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides,
Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor
miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium
purpurogenum, Phanerochaete chrysosporium, Phlebia radiata,
Pleurotus eryngii, Talaromyces emersonii, Thielavia terrestris,
Trametes villosa, Trametes versicolor, Trichoderma harzianum,
Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma
reesei, or Trichoderma viride cell.
[0316] Fungal cells may be transformed by a process involving
protoplast formation, transformation of the protoplasts, and
regeneration of the cell wall in a manner known per se. Suitable
procedures for transformation of Aspergillus and Trichoderma host
cells are described in EP 238023, Yelton et al., 1984, Proc. Natl.
Acad. Sci. USA 81: 1470-1474, and Christensen et al., 1988,
Bio/Technology 6: 1419-1422. Suitable methods for transforming
Fusarium species are described by Malardier et al., 1989, Gene 78:
147-156, and WO 96/00787. Yeast may be transformed using the
procedures described by Becker and Guarente, In Abelson, J. N. and
Simon, M. I., editors, Guide to Yeast Genetics and Molecular
Biology, Methods in Enzymology, Volume 194, pp. 182-187, Academic
Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153: 163;
and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.
Methods of Production
[0317] The present invention also relates to methods of producing a
variant, comprising (a) cultivating a recombinant host cell of the
present invention under conditions conducive for production of the
variant; and optionally (b) recovering the variant.
[0318] The host cells are cultivated in a nutrient medium suitable
for production of the variant using methods known in the art. For
example, the cells may be cultivated by shake flask cultivation, or
small-scale or large-scale fermentation (including continuous,
batch, fed-batch, or solid state fermentations) in laboratory or
industrial fermentors in a suitable medium and under conditions
allowing the variant to be expressed and/or isolated. The
cultivation takes place in a suitable nutrient medium comprising
carbon and nitrogen sources and inorganic salts, using procedures
known in the art. Suitable media are available from commercial
suppliers or may be prepared according to published compositions
(e.g., in catalogues of the American Type Culture Collection). If
the variant is secreted into the nutrient medium, the variant can
be recovered directly from the medium. If the variant is not
secreted, it can be recovered from cell lysates.
[0319] The variants may be detected using methods known in the art
that are specific for the variants. These detection methods
include, but are not limited to, use of specific antibodies,
formation of an enzyme product, or disappearance of an enzyme
substrate. For example, an enzyme assay may be used to determine
the activity of the variant.
[0320] The variant may be recovered using methods known in the art.
For example, the variant may be recovered from the nutrient medium
by conventional procedures including, but not limited to,
collection, centrifugation, filtration, extraction, spray-drying,
evaporation, or precipitation. In one aspect, a whole fermentation
broth comprising a variant of the present invention is
recovered.
[0321] The variant may be purified by a variety of procedures known
in the art including, but not limited to, chromatography (e.g., ion
exchange, affinity, hydrophobic, chromatofocusing, and size
exclusion), electrophoretic procedures (e.g., preparative
isoelectric focusing), differential solubility (e.g., ammonium
sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein
Purification, Janson and Ryden, editors, VCH Publishers, New York,
1989) to obtain substantially pure variants.
Fermentation Broth Formulations or Cell Compositions
[0322] The present invention also relates to a fermentation broth
formulation or a cell composition comprising a variant of the
present invention. The fermentation broth product further comprises
additional ingredients used in the fermentation process, such as,
for example, cells (including, the host cells containing the gene
encoding the variant of the present invention which are used to
produce the variant), cell debris, biomass, fermentation media
and/or fermentation products. In some embodiments, the composition
is a cell-killed whole broth containing organic acid(s), killed
cells and/or cell debris, and culture medium.
[0323] The term "fermentation broth" as used herein refers to a
preparation produced by cellular fermentation that undergoes no or
minimal recovery and/or purification. For example, fermentation
broths are produced when microbial cultures are grown to
saturation, incubated under carbon-limiting conditions to allow
protein synthesis (e.g., expression of enzymes by host cells) and
secretion into cell culture medium. The fermentation broth can
contain unfractionated or fractionated contents of the fermentation
materials derived at the end of the fermentation. Typically, the
fermentation broth is unfractionated and comprises the spent
culture medium and cell debris present after the microbial cells
(e.g., filamentous fungal cells) are removed, e.g., by
centrifugation. In some embodiments, the fermentation broth
contains spent cell culture medium, extracellular enzymes, and
viable and/or nonviable microbial cells.
[0324] In an embodiment, the fermentation broth formulation and
cell compositions comprise a first organic acid component
comprising at least one 1-5 carbon organic acid and/or a salt
thereof and a second organic acid component comprising at least one
6 or more carbon organic acid and/or a salt thereof. In a specific
embodiment, the first organic acid component is acetic acid, formic
acid, propionic acid, a salt thereof, or a mixture of two or more
of the foregoing and the second organic acid component is benzoic
acid, cyclohexanecarboxylic acid, 4-methylvaleric acid,
phenylacetic acid, a salt thereof, or a mixture of two or more of
the foregoing.
[0325] In one aspect, the composition contains an organic acid(s),
and optionally further contains killed cells and/or cell debris. In
one embodiment, the killed cells and/or cell debris are removed
from a cell-killed whole broth to provide a composition that is
free of these components.
[0326] The fermentation broth formulations or cell compositions may
further comprise a preservative and/or anti-microbial (e.g.,
bacteriostatic) agent, including, but not limited to, sorbitol,
sodium chloride, potassium sorbate, and others known in the
art.
[0327] The fermentation broth formulations or cell compositions may
further comprise multiple enzymatic activities, such as one or more
(e.g., several) enzymes selected from the group consisting of a
cellulase, a hemicellulase, a catalase, an esterase, an expansin, a
laccase, a ligninolytic enzyme, a pectinase, a peroxidase, a
protease, and a swollenin. The fermentation broth formulations or
cell compositions may also comprise one or more (e.g., several)
enzymes selected from the group consisting of a hydrolase, an
isomerase, a ligase, a lyase, an oxidoreductase, or a transferase,
e.g., an alpha-galactosidase, alpha-glucosidase, aminopeptidase,
amylase, beta-galactosidase, beta-glucosidase, beta-xylosidase,
carbohydrase, carboxypeptidase, catalase, cellobiohydrolase,
cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase,
deoxyribonuclease, endoglucanase, esterase, glucoamylase,
invertase, laccase, lipase, mannosidase, mutanase, oxidase,
pectinolytic enzyme, peroxidase, phytase, polyphenoloxidase,
proteolytic enzyme, ribonuclease, transglutaminase, or
xylanase.
[0328] The cell-killed whole broth or composition may contain the
unfractionated contents of the fermentation materials derived at
the end of the fermentation. Typically, the cell-killed whole broth
or composition contains the spent culture medium and cell debris
present after the microbial cells (e.g., filamentous fungal cells)
are grown to saturation, incubated under carbon-limiting conditions
to allow protein synthesis (e.g., expression of cellulase and/or
glucosidase enzyme(s)). In some embodiments, the cell-killed whole
broth or composition contains the spent cell culture medium,
extracellular enzymes, and killed filamentous fungal cells. In some
embodiments, the microbial cells present in the cell-killed whole
broth or composition can be permeabilized and/or lysed using
methods known in the art.
[0329] A whole broth or cell composition as described herein is
typically a liquid, but may contain insoluble components, such as
killed cells, cell debris, culture media components, and/or
insoluble enzyme(s). In some embodiments, insoluble components may
be removed to provide a clarified liquid composition.
[0330] The whole broth formulations and cell compositions of the
present invention may be produced by a method described in WO
90/15861 or WO 2010/096673.
[0331] Examples are given below of preferred uses of the
compositions of the present invention. The dosage of the
composition and other conditions under which the composition is
used may be determined on the basis of methods known in the
art.
Enzyme Compositions
[0332] The present invention also relates to compositions
comprising a variant of the present invention. Preferably, the
compositions are enriched in such a variant. The term "enriched"
indicates that the cellobiohydrolase activity of the composition
has been increased, e.g., with an enrichment factor of at least
1.1.
[0333] The compositions may comprise a variant of the present
invention as the major enzymatic component, e.g., a mono-component
composition. Alternatively, the compositions may comprise multiple
enzymatic activities, such as one or more (e.g., several) enzymes
selected from the group consisting of a cellulase, a hemicellulase,
a GH61 polypeptide having cellulolytic enhancing activity, a
catalase, an esterase, an expansin, a laccase, a ligninolytic
enzyme, a pectinase, a peroxidase, a protease, and a swollenin. The
compositions may also comprise one or more (e.g., several) enzymes
selected from the group consisting of a hydrolase, an isomerase, a
ligase, a lyase, an oxidoreductase, or a transferase, e.g., an
alpha-galactosidase, alpha-glucosidase, aminopeptidase, amylase,
beta-galactosidase, beta-glucosidase, beta-xylosidase,
carbohydrase, carboxypeptidase, catalase, cellobiohydrolase,
cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase,
deoxyribonuclease, endoglucanase, esterase, glucoamylase,
invertase, laccase, lipase, mannosidase, mutanase, oxidase,
pectinolytic enzyme, peroxidase, phytase, polyphenoloxidase,
proteolytic enzyme, ribonuclease, transglutaminase, or xylanase.
The compositions may be prepared in accordance with methods known
in the art and may be in the form of a liquid or a dry composition.
The compositions may be stabilized in accordance with methods known
in the art.
[0334] Examples are given below of preferred uses of the
compositions of the present invention. The dosage of the
composition and other conditions under which the composition is
used may be determined on the basis of methods known in the
art.
Uses
[0335] The present invention is also directed to the following
processes for using the variants having cellobiohydrolase I
activity of the present invention, or compositions thereof. The
present invention also relates to processes for degrading a
cellulosic material, comprising: treating the cellulosic material
with an enzyme composition in the presence of a cellobiohydrolase
variant of the present invention. In one aspect, the processes
further comprise recovering the degraded cellulosic material.
Soluble products of degradation of the cellulosic material can be
separated from insoluble cellulosic material using a method known
in the art such as, for example, centrifugation, filtration, or
gravity settling.
[0336] The present invention also relates to processes of producing
a fermentation product, comprising: (a) saccharifying a cellulosic
material with an enzyme composition in the presence of a
cellobiohydrolase variant of the present invention; (b) fermenting
the saccharified cellulosic material with one or more (e.g.,
several) fermenting microorganisms to produce the fermentation
product; and (c) recovering the fermentation product from the
fermentation.
[0337] The present invention also relates to processes of
fermenting a cellulosic material, comprising: fermenting the
cellulosic material with one or more (e.g., several) fermenting
microorganisms, wherein the cellulosic material is saccharified
with an enzyme composition in the presence of a cellobiohydrolase
variant of the present invention. In one aspect, the fermenting of
the cellulosic material produces a fermentation product. In another
aspect, the processes further comprise recovering the fermentation
product from the fermentation.
[0338] The processes of the present invention can be used to
saccharify the cellulosic material to fermentable sugars and to
convert the fermentable sugars to many useful fermentation
products, e.g., fuel (ethanol, n-butanol, isobutanol, biodiesel,
jet fuel) and/or platform chemicals (e.g., acids, alcohols,
ketones, gases, oils, and the like). The production of a desired
fermentation product from the cellulosic material typically
involves pretreatment, enzymatic hydrolysis (saccharification), and
fermentation.
[0339] The processing of the cellulosic material according to the
present invention can be accomplished using methods conventional in
the art. Moreover, the processes of the present invention can be
implemented using any conventional biomass processing apparatus
configured to operate in accordance with the invention.
[0340] Hydrolysis (saccharification) and fermentation, separate or
simultaneous, include, but are not limited to, separate hydrolysis
and fermentation (SHF); simultaneous saccharification and
fermentation (SSF); simultaneous saccharification and
co-fermentation (SSCF); hybrid hydrolysis and fermentation (HHF);
separate hydrolysis and co-fermentation (SHCF); hybrid hydrolysis
and co-fermentation (HHCF); and direct microbial conversion (DMC),
also sometimes called consolidated bioprocessing (CBP). SHF uses
separate process steps to first enzymatically hydrolyze the
cellulosic material to fermentable sugars, e.g., glucose,
cellobiose, and pentose monomers, and then ferment the fermentable
sugars to ethanol. In SSF, the enzymatic hydrolysis of the
cellulosic material and the fermentation of sugars to ethanol are
combined in one step (Philippidis, G. P., 1996, Cellulose
bioconversion technology, in Handbook on Bioethanol: Production and
Utilization, Wyman, C. E., ed., Taylor & Francis, Washington,
D.C., 179-212). SSCF involves the co-fermentation of multiple
sugars (Sheehan and Himmel, 1999, Biotechnol. Prog. 15: 817-827).
HHF involves a separate hydrolysis step, and in addition a
simultaneous saccharification and hydrolysis step, which can be
carried out in the same reactor. The steps in an HHF process can be
carried out at different temperatures, i.e., high temperature
enzymatic saccharification followed by SSF at a lower temperature
that the fermentation strain can tolerate. DMC combines all three
processes (enzyme production, hydrolysis, and fermentation) in one
or more (e.g., several) steps where the same organism is used to
produce the enzymes for conversion of the cellulosic material to
fermentable sugars and to convert the fermentable sugars into a
final product (Lynd et al., 2002, Microbiol. Mol. Biol. Reviews 66:
506-577). It is understood herein that any method known in the art
comprising pretreatment, enzymatic hydrolysis (saccharification),
fermentation, or a combination thereof, can be used in the
practicing the processes of the present invention.
[0341] A conventional apparatus can include a fed-batch stirred
reactor, a batch stirred reactor, a continuous flow stirred reactor
with ultrafiltration, and/or a continuous plug-flow column reactor
(de Castilhos Corazza et al., 2003, Acta Scientiarum. Technology
25: 33-38; Gusakov and Sinitsyn, 1985, Enz. Microb. Technol. 7:
346-352), an attrition reactor (Ryu and Lee, 1983, Biotechnol.
Bioeng. 25: 53-65). Additional reactor types include fluidized bed,
upflow blanket, immobilized, and extruder type reactors for
hydrolysis and/or fermentation.
[0342] Pretreatment.
[0343] In practicing the processes of the present invention, any
pretreatment process known in the art can be used to disrupt plant
cell wall components of the cellulosic material (Chandra et al.,
2007, Adv. Biochem. Engin./Biotechnol. 108: 67-93; Galbe and
Zacchi, 2007, Adv. Biochem. Engin./Biotechnol. 108: 41-65; Hendriks
and Zeeman, 2009, Bioresource Technol. 100: 10-18; Mosier et al.,
2005, Bioresource Technol. 96: 673-686; Taherzadeh and Karimi,
2008, Int. J. Mol. Sci. 9: 1621-1651; Yang and Wyman, 2008,
Biofuels Bioproducts and Biorefining-Biofpr. 2: 26-40).
[0344] The cellulosic material can also be subjected to particle
size reduction, sieving, pre-soaking, wetting, washing, and/or
conditioning prior to pretreatment using methods known in the
art.
[0345] Conventional pretreatments include, but are not limited to,
steam pretreatment (with or without explosion), dilute acid
pretreatment, hot water pretreatment, alkaline pretreatment, lime
pretreatment, wet oxidation, wet explosion, ammonia fiber
explosion, organosolv pretreatment, and biological pretreatment.
Additional pretreatments include ammonia percolation, ultrasound,
electroporation, microwave, supercritical CO.sub.2, supercritical
H.sub.2O, ozone, ionic liquid, and gamma irradiation
pretreatments.
[0346] The cellulosic material can be pretreated before hydrolysis
and/or fermentation. Pretreatment is preferably performed prior to
the hydrolysis. Alternatively, the pretreatment can be carried out
simultaneously with enzyme hydrolysis to release fermentable
sugars, such as glucose, xylose, and/or cellobiose. In most cases
the pretreatment step itself results in some conversion of biomass
to fermentable sugars (even in absence of enzymes).
[0347] Steam Pretreatment.
[0348] In steam pretreatment, the cellulosic material is heated to
disrupt the plant cell wall components, including lignin,
hemicellulose, and cellulose to make the cellulose and other
fractions, e.g., hemicellulose, accessible to enzymes. The
cellulosic material is passed to or through a reaction vessel where
steam is injected to increase the temperature to the required
temperature and pressure and is retained therein for the desired
reaction time. Steam pretreatment is preferably performed at
140-250.degree. C., e.g., 160-200.degree. C. or 170-190.degree. C.,
where the optimal temperature range depends on optional addition of
a chemical catalyst. Residence time for the steam pretreatment is
preferably 1-60 minutes, e.g., 1-30 minutes, 1-20 minutes, 3-12
minutes, or 4-10 minutes, where the optimal residence time depends
on the temperature and optional addition of a chemical catalyst.
Steam pretreatment allows for relatively high solids loadings, so
that the cellulosic material is generally only moist during the
pretreatment. The steam pretreatment is often combined with an
explosive discharge of the material after the pretreatment, which
is known as steam explosion, that is, rapid flashing to atmospheric
pressure and turbulent flow of the material to increase the
accessible surface area by fragmentation (Duff and Murray, 1996,
Bioresource Technology 855: 1-33; Galbe and Zacchi, 2002, Appl.
Microbiol. Biotechnol. 59: 618-628; U.S. Patent Application No.
2002/0164730). During steam pretreatment, hemicellulose acetyl
groups are cleaved and the resulting acid autocatalyzes partial
hydrolysis of the hemicellulose to monosaccharides and
oligosaccharides. Lignin is removed to only a limited extent.
[0349] Chemical Pretreatment:
[0350] The term "chemical treatment" refers to any chemical
pretreatment that promotes the separation and/or release of
cellulose, hemicellulose, and/or lignin. Such a pretreatment can
convert crystalline cellulose to amorphous cellulose. Examples of
suitable chemical pretreatment processes include, for example,
dilute acid pretreatment, lime pretreatment, wet oxidation, ammonia
fiber/freeze expansion (AFEX), ammonia percolation (APR), ionic
liquid, and organosolv pretreatments.
[0351] A chemical catalyst such as H.sub.2SO.sub.4 or SO.sub.2
(typically 0.3 to 5% w/w) is sometimes added prior to steam
pretreatment, which decreases the time and temperature, increases
the recovery, and improves enzymatic hydrolysis (Ballesteros et
al., 2006, Appl. Biochem. Biotechnol. 129-132: 496-508; Varga et
al., 2004, Appl. Biochem. Biotechnol. 113-116: 509-523; Sassner et
al., 2006, Enzyme Microb. Technol. 39: 756-762). In dilute acid
pretreatment, the cellulosic material is mixed with dilute acid,
typically H.sub.2SO.sub.4, and water to form a slurry, heated by
steam to the desired temperature, and after a residence time
flashed to atmospheric pressure. The dilute acid pretreatment can
be performed with a number of reactor designs, e.g., plug-flow
reactors, counter-current reactors, or continuous counter-current
shrinking bed reactors (Duff and Murray, 1996, supra; Schell et
al., 2004, Bioresource Technology 91: 179-188; Lee et al., 1999,
Adv. Biochem. Eng. Biotechnol. 65: 93-115).
[0352] Several methods of pretreatment under alkaline conditions
can also be used. These alkaline pretreatments include, but are not
limited to, sodium hydroxide, lime, wet oxidation, ammonia
percolation (APR), and ammonia fiber/freeze expansion (AFEX)
pretreatment.
[0353] Lime pretreatment is performed with calcium oxide or calcium
hydroxide at temperatures of 85-150.degree. C. and residence times
from 1 hour to several days (Wyman et al., 2005, Bioresource
Technol. 96: 1959-1966; Mosier et al., 2005, Bioresource Technol.
96: 673-686). WO 2006/110891, WO 2006/110899, WO 2006/110900, and
WO 2006/110901 disclose pretreatment methods using ammonia.
[0354] Wet oxidation is a thermal pretreatment performed typically
at 180-200.degree. C. for 5-15 minutes with addition of an
oxidative agent such as hydrogen peroxide or over-pressure of
oxygen (Schmidt and Thomsen, 1998, Bioresource Technol. 64:
139-151; Palonen et al., 2004, Appl. Biochem. Biotechnol. 117:
1-17; Varga et al., 2004, Biotechnol. Bioeng. 88: 567-574; Martin
et al., 2006, J. Chem. Technol. Biotechnol. 81: 1669-1677). The
pretreatment is performed preferably at 1-40% dry matter, e.g.,
2-30% dry matter or 5-20% dry matter, and often the initial pH is
increased by the addition of alkali such as sodium carbonate.
[0355] A modification of the wet oxidation pretreatment method,
known as wet explosion (combination of wet oxidation and steam
explosion) can handle dry matter up to 30%. In wet explosion, the
oxidizing agent is introduced during pretreatment after a certain
residence time. The pretreatment is then ended by flashing to
atmospheric pressure (WO 2006/032282).
[0356] Ammonia fiber expansion (AFEX) involves treating the
cellulosic material with liquid or gaseous ammonia at moderate
temperatures such as 90-150.degree. C. and high pressure such as
17-20 bar for 5-10 minutes, where the dry matter content can be as
high as 60% (Gollapalli et al., 2002, Appl. Biochem. Biotechnol.
98: 23-35; Chundawat et al., 2007, Biotechnol. Bioeng. 96: 219-231;
Alizadeh et al., 2005, Appl. Biochem. Biotechnol. 121: 1133-1141;
Teymouri et al., 2005, Bioresource Technology 96: 2014-2018).
During AFEX pretreatment cellulose and hemicelluloses remain
relatively intact. Lignin-carbohydrate complexes are cleaved.
[0357] Organosolv pretreatment delignifies the cellulosic material
by extraction using aqueous ethanol (40-60% ethanol) at
160-200.degree. C. for 30-60 minutes (Pan et al., 2005, Biotechnol.
Bioeng. 90: 473-481; Pan et al., 2006, Biotechnol. Bioeng. 94:
851-861; Kurabi et al., 2005, Appl. Biochem. Biotechnol. 121:
219-230). Sulphuric acid is usually added as a catalyst. In
organosolv pretreatment, the majority of hemicellulose and lignin
is removed.
[0358] Other examples of suitable pretreatment methods are
described by Schell et al., 2003, Appl. Biochem. Biotechnol.
105-108: 69-85, and Mosier et al., 2005, Bioresource Technology 96:
673-686, and U.S. Published Application 2002/0164730.
[0359] In one aspect, the chemical pretreatment is preferably
carried out as a dilute acid treatment, and more preferably as a
continuous dilute acid treatment. The acid is typically sulfuric
acid, but other acids can also be used, such as acetic acid, citric
acid, nitric acid, phosphoric acid, tartaric acid, succinic acid,
hydrogen chloride, or mixtures thereof. Mild acid treatment is
conducted in the pH range of preferably 1-5, e.g., 1-4 or 1-2.5. In
one aspect, the acid concentration is in the range from preferably
0.01 to 10 wt. % acid, e.g., 0.05 to 5 wt. % acid or 0.1 to 2 wt. %
acid. The acid is contacted with the cellulosic material and held
at a temperature in the range of preferably 140-200.degree. C.,
e.g., 165-190.degree. C., for periods ranging from 1 to 60
minutes.
[0360] In another aspect, pretreatment takes place in an aqueous
slurry. In preferred aspects, the cellulosic material is present
during pretreatment in amounts preferably between 10-80 wt %, e.g.,
20-70 wt % or 30-60 wt %, such as around 40 wt %. The pretreated
cellulosic material can be unwashed or washed using any method
known in the art, e.g., washed with water.
[0361] Mechanical Pretreatment or Physical Pretreatment: The term
"mechanical pretreatment" or "physical pretreatment" refers to any
pretreatment that promotes size reduction of particles. For
example, such pretreatment can involve various types of grinding or
milling (e.g., dry milling, wet milling, or vibratory ball
milling).
[0362] The cellulosic material can be pretreated both physically
(mechanically) and chemically. Mechanical or physical pretreatment
can be coupled with steaming/steam explosion, hydrothermolysis,
dilute or mild acid treatment, high temperature, high pressure
treatment, irradiation (e.g., microwave irradiation), or
combinations thereof. In one aspect, high pressure means pressure
in the range of preferably about 100 to about 400 psi, e.g., about
150 to about 250 psi. In another aspect, high temperature means
temperature in the range of about 100 to about 300.degree. C.,
e.g., about 140 to about 200.degree. C. In a preferred aspect,
mechanical or physical pretreatment is performed in a batch-process
using a steam gun hydrolyzer system that uses high pressure and
high temperature as defined above, e.g., a Sunds Hydrolyzer
available from Sunds Defibrator AB, Sweden. The physical and
chemical pretreatments can be carried out sequentially or
simultaneously, as desired.
[0363] Accordingly, in a preferred aspect, the cellulosic material
is subjected to physical (mechanical) or chemical pretreatment, or
any combination thereof, to promote the separation and/or release
of cellulose, hemicellulose, and/or lignin.
[0364] Biological Pretreatment: The term "biological pretreatment"
refers to any biological pretreatment that promotes the separation
and/or release of cellulose, hemicellulose, and/or lignin from the
cellulosic material. Biological pretreatment techniques can involve
applying lignin-solubilizing microorganisms and/or enzymes (see,
for example, Hsu, T.-A., 1996, Pretreatment of biomass, in Handbook
on Bioethanol: Production and Utilization, Wyman, C. E., ed.,
Taylor & Francis, Washington, D.C., 179-212; Ghosh and Singh,
1993, Adv. Appl. Microbiol. 39: 295-333; McMillan, J. D., 1994,
Pretreating lignocellulosic biomass: a review, in Enzymatic
Conversion of Biomass for Fuels Production, Himmel, M. E., Baker,
J. O., and Overend, R. P., eds., ACS Symposium Series 566, American
Chemical Society, Washington, D.C., chapter 15; Gong, C. S., Cao,
N. J., Du, J., and Tsao, G. T., 1999, Ethanol production from
renewable resources, in Advances in Biochemical
Engineering/Biotechnology, Scheper, T., ed., Springer-Verlag Berlin
Heidelberg, Germany, 65: 207-241; Olsson and Hahn-Hagerdal, 1996,
Enz. Microb. Tech. 18: 312-331; and Vallander and Eriksson, 1990,
Adv. Biochem. Eng./Biotechnol. 42: 63-95).
[0365] Saccharification.
[0366] In the hydrolysis step, also known as saccharification, the
cellulosic material, e.g., pretreated, is hydrolyzed to break down
cellulose and/or hemicellulose to fermentable sugars, such as
glucose, cellobiose, xylose, xylulose, arabinose, mannose,
galactose, and/or soluble oligosaccharides. The hydrolysis is
performed enzymatically by an enzyme composition in the presence of
a cellobiohydrolase variant of the present invention. The enzymes
of the compositions can be added simultaneously or
sequentially.
[0367] Enzymatic hydrolysis is preferably carried out in a suitable
aqueous environment under conditions that can be readily determined
by one skilled in the art. In one aspect, hydrolysis is performed
under conditions suitable for the activity of the enzymes(s), i.e.,
optimal for the enzyme(s). The hydrolysis can be carried out as a
fed batch or continuous process where the cellulosic material is
fed gradually to, for example, an enzyme containing hydrolysis
solution.
[0368] The saccharification is generally performed in stirred-tank
reactors or fermentors under controlled pH, temperature, and mixing
conditions. Suitable process time, temperature and pH conditions
can readily be determined by one skilled in the art. For example,
the saccharification can last up to 200 hours, but is typically
performed for preferably about 12 to about 120 hours, e.g., about
16 to about 72 hours or about 24 to about 48 hours. The temperature
is in the range of preferably about 25.degree. C. to about
70.degree. C., e.g., about 30.degree. C. to about 65.degree. C.,
about 40.degree. C. to about 60.degree. C., or about 50.degree. C.
to about 55.degree. C. The pH is in the range of preferably about 3
to about 8, e.g., about 3.5 to about 7, about 4 to about 6, or
about 4.5 to about 5.5. The dry solids content is in the range of
preferably about 5 to about 50 wt %, e.g., about 10 to about 40 wt
% or about 20 to about 30 wt %.
[0369] The enzyme compositions can comprise any protein useful in
degrading the cellulosic material.
[0370] In one aspect, the enzyme composition comprises or further
comprises one or more (e.g., several) proteins selected from the
group consisting of a cellulase, a GH61 polypeptide having
cellulolytic enhancing activity, a hemicellulase, an esterase, an
expansin, a ligninolytic enzyme, an oxidoreductase, a pectinase, a
protease, and a swollenin. In another aspect, the cellulase is
preferably one or more (e.g., several) enzymes selected from the
group consisting of an endoglucanase, a cellobiohydrolase, and a
beta-glucosidase. In another aspect, the hemicellulase is
preferably one or more (e.g., several) enzymes selected from the
group consisting of an acetylmannan esterase, an acetylxylan
esterase, an arabinanase, an arabinofuranosidase, a coumaric acid
esterase, a feruloyl esterase, a galactosidase, a glucuronidase, a
glucuronoyl esterase, a mannanase, a mannosidase, a xylanase, and a
xylosidase. In another aspect, the oxidoreductase is preferably one
or more (e.g., several) enzymes selected from the group consisting
of a catalase, a laccase, and a peroxidase.
[0371] In another aspect, the enzyme composition comprises one or
more (e.g., several) cellulolytic enzymes. In another aspect, the
enzyme composition comprises or further comprises one or more
(e.g., several) hemicellulolytic enzymes. In another aspect, the
enzyme composition comprises one or more (e.g., several)
cellulolytic enzymes and one or more (e.g., several)
hemicellulolytic enzymes. In another aspect, the enzyme composition
comprises one or more (e.g., several) enzymes selected from the
group of cellulolytic enzymes and hemicellulolytic enzymes. In
another aspect, the enzyme composition comprises an endoglucanase.
In another aspect, the enzyme composition comprises a
cellobiohydrolase. In another aspect, the enzyme composition
comprises a beta-glucosidase. In another aspect, the enzyme
composition comprises a GH61 polypeptide having cellulolytic
enhancing activity. In another aspect, the enzyme composition
comprises an endoglucanase and a GH61 polypeptide having
cellulolytic enhancing activity. In another aspect, the enzyme
composition comprises a cellobiohydrolase and a GH61 polypeptide
having cellulolytic enhancing activity. In another aspect, the
enzyme composition comprises a beta-glucosidase and a GH61
polypeptide having cellulolytic enhancing activity. In another
aspect, the enzyme composition comprises an endoglucanase and a
cellobiohydrolase. In another aspect, the enzyme composition
comprises an endoglucanase and a cellobiohydrolase I, a
cellobiohydrolase II, or a combination of a cellobiohydrolase I and
a cellobiohydrolase II. In another aspect, the enzyme composition
comprises an endoglucanase and a beta-glucosidase. In another
aspect, the enzyme composition comprises a beta-glucosidase and a
cellobiohydrolase. In another aspect, the enzyme composition
comprises a beta-glucosidase and a cellobiohydrolase I, a
cellobiohydrolase II, or a combination of a cellobiohydrolase I and
a cellobiohydrolase II In another aspect, the enzyme composition
comprises an endoglucanase, a GH61 polypeptide having cellulolytic
enhancing activity, and a cellobiohydrolase. In another aspect, the
enzyme composition comprises an endoglucanase, a GH61 polypeptide
having cellulolytic enhancing activity, and a cellobiohydrolase I,
a cellobiohydrolase II, or a combination of a cellobiohydrolase I
and a cellobiohydrolase II. In another aspect, the enzyme
composition comprises an endoglucanase, a beta-glucosidase, and a
GH61 polypeptide having cellulolytic enhancing activity. In another
aspect, the enzyme composition comprises a beta-glucosidase, a GH61
polypeptide having cellulolytic enhancing activity, and a
cellobiohydrolase. In another aspect, the enzyme composition
comprises a beta-glucosidase, a GH61 polypeptide having
cellulolytic enhancing activity, and a cellobiohydrolase I, a
cellobiohydrolase II, or a combination of a cellobiohydrolase I and
a cellobiohydrolase II. In another aspect, the enzyme composition
comprises an endoglucanase, a beta-glucosidase, and a
cellobiohydrolase. In another aspect, the enzyme composition
comprises an endoglucanase, a beta-glucosidase, and a
cellobiohydrolase I, a cellobiohydrolase II, or a combination of a
cellobiohydrolase I and a cellobiohydrolase II. In another aspect,
the enzyme composition comprises an endoglucanase, a
cellobiohydrolase, a beta-glucosidase, and a GH61 polypeptide
having cellulolytic enhancing activity. In another aspect, the
enzyme composition comprises an endoglucanase, a beta-glucosidase,
a GH61 polypeptide having cellulolytic enhancing activity, and a
cellobiohydrolase I, a cellobiohydrolase II, or a combination of a
cellobiohydrolase I and a cellobiohydrolase II.
[0372] In another aspect, the enzyme composition comprises an
acetylmannan esterase. In another aspect, the enzyme composition
comprises an acetylxylan esterase. In another aspect, the enzyme
composition comprises an arabinanase (e.g., alpha-L-arabinanase).
In another aspect, the enzyme composition comprises an
arabinofuranosidase (e.g., alpha-L-arabinofuranosidase). In another
aspect, the enzyme composition comprises a coumaric acid esterase.
In another aspect, the enzyme composition comprises a feruloyl
esterase. In another aspect, the enzyme composition comprises a
galactosidase (e.g., alpha-galactosidase and/or
beta-galactosidase). In another aspect, the enzyme composition
comprises a glucuronidase (e.g., alpha-D-glucuronidase). In another
aspect, the enzyme composition comprises a glucuronoyl esterase. In
another aspect, the enzyme composition comprises a mannanase. In
another aspect, the enzyme composition comprises a mannosidase
(e.g., beta-mannosidase). In another aspect, the enzyme composition
comprises a xylanase. In a preferred aspect, the xylanase is a
Family 10 xylanase. In another preferred aspect, the xylanase is a
Family 11 xylanase. In another aspect, the enzyme composition
comprises a xylosidase (e.g., beta-xylosidase).
[0373] In another aspect, the enzyme composition comprises an
esterase. In another aspect, the enzyme composition comprises an
expansin. In another aspect, the enzyme composition comprises a
ligninolytic enzyme. In a preferred aspect, the ligninolytic enzyme
is a manganese peroxidase. In another preferred aspect, the
ligninolytic enzyme is a lignin peroxidase. In another preferred
aspect, the ligninolytic enzyme is a H.sub.2O.sub.2-producing
enzyme. In another aspect, the enzyme composition comprises a
pectinase. In another aspect, the enzyme composition comprises an
oxidoreductase. In another preferred aspect, the oxidoreductase is
a catalase. In another preferred aspect, the oxidoreductase is a
laccase. In another preferred aspect, the oxidoreductase is a
peroxidase. In another aspect, the enzyme composition comprises a
protease. In another aspect, the enzyme composition comprises a
swollenin.
[0374] In the processes of the present invention, the enzyme(s) can
be added prior to or during saccharification, saccharification and
fermentation, or fermentation.
[0375] One or more (e.g., several) components of the enzyme
composition may be native proteins, recombinant proteins, or a
combination of native proteins and recombinant proteins. For
example, one or more (e.g., several) components may be native
proteins of a cell, which is used as a host cell to express
recombinantly one or more (e.g., several) other components of the
enzyme composition. It is understood herein that the recombinant
proteins may be heterologous (e.g., foreign) and native to the host
cell. One or more (e.g., several) components of the enzyme
composition may be produced as monocomponents, which are then
combined to form the enzyme composition. The enzyme composition may
be a combination of multicomponent and monocomponent protein
preparations.
[0376] The enzymes used in the processes of the present invention
may be in any form suitable for use, such as, for example, a
fermentation broth formulation or a cell composition, a cell lysate
with or without cellular debris, a semi-purified or purified enzyme
preparation, or a host cell as a source of the enzymes. The enzyme
composition may be a dry powder or granulate, a non-dusting
granulate, a liquid, a stabilized liquid, or a stabilized protected
enzyme. Liquid enzyme preparations may, for instance, be stabilized
by adding stabilizers such as a sugar, a sugar alcohol or another
polyol, and/or lactic acid or another organic acid according to
established processes.
[0377] The optimum amounts of the enzymes and the cellobiohydrolase
variant depend on several factors including, but not limited to,
the mixture of component cellulolytic enzymes and/or
hemicellulolytic enzymes, the cellulosic material, the
concentration of cellulosic material, the pretreatment(s) of the
cellulosic material, temperature, time, pH, and inclusion of
fermenting organism (e.g., for Simultaneous Saccharification and
Fermentation).
[0378] In one aspect, an effective amount of cellulolytic or
hemicellulolytic enzyme to the cellulosic material is about 0.5 to
about 50 mg, e.g., about 0.5 to about 40 mg, about 0.5 to about 25
mg, about 0.75 to about 20 mg, about 0.75 to about 15 mg, about 0.5
to about 10 mg, or about 2.5 to about 10 mg per g of the cellulosic
material.
[0379] In another aspect, an effective amount of a
cellobiohydrolase variant to the cellulosic material is about 0.01
to about 50.0 mg, e.g., about 0.01 to about 40 mg, about 0.01 to
about 30 mg, about 0.01 to about 20 mg, about 0.01 to about 10 mg,
about 0.01 to about 5 mg, about 0.025 to about 1.5 mg, about 0.05
to about 1.25 mg, about 0.075 to about 1.25 mg, about 0.1 to about
1.25 mg, about 0.15 to about 1.25 mg, or about 0.25 to about 1.0 mg
per g of the cellulosic material.
[0380] In another aspect, an effective amount of a
cellobiohydrolase variant to cellulolytic or hemicellulolytic
enzyme is about 0.005 to about 1.0 g, e.g., about 0.01 to about 1.0
g, about 0.15 to about 0.75 g, about 0.15 to about 0.5 g, about 0.1
to about 0.5 g, about 0.1 to about 0.25 g, or about 0.05 to about
0.2 g per g of cellulolytic or hemicellulolytic enzyme.
[0381] The polypeptides having cellulolytic enzyme activity or
hemicellulolytic enzyme activity as well as other
proteins/polypeptides useful in the degradation of the cellulosic
material, e.g., GH61 polypeptides having cellulolytic enhancing
activity, (collectively hereinafter "polypeptides having enzyme
activity") can be derived or obtained from any suitable origin,
including, archaeal, bacterial, fungal, yeast, plant, or animal
origin. The term "obtained" also means herein that the enzyme may
have been produced recombinantly in a host organism employing
methods described herein, wherein the recombinantly produced enzyme
is either native or foreign to the host organism or has a modified
amino acid sequence, e.g., having one or more (e.g., several) amino
acids that are deleted, inserted and/or substituted, i.e., a
recombinantly produced enzyme that is a mutant and/or a fragment of
a native amino acid sequence or an enzyme produced by nucleic acid
shuffling processes known in the art. Encompassed within the
meaning of a native enzyme are natural variants and within the
meaning of a foreign enzyme are variants obtained by, for example,
site-directed mutagenesis or shuffling.
[0382] A polypeptide having enzyme activity may be a bacterial
polypeptide. For example, the polypeptide may be a Gram positive
bacterial polypeptide such as a Bacillus, Streptococcus,
Streptomyces, Staphylococcus, Enterococcus, Lactobacillus,
Lactococcus, Clostridium, Geobacillus, Caldicellulosiruptor,
Acidothermus, Thermobifidia, or Oceanobacillus polypeptide having
enzyme activity, or a Gram negative bacterial polypeptide such as
an E. coli, Pseudomonas, Salmonella, Campylobacter, Helicobacter,
Flavobacterium, Fusobacterium, Ilyobacter, Neisseria, or Ureaplasma
polypeptide having enzyme activity.
[0383] In one aspect, the polypeptide is a Bacillus alkalophilus,
Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans,
Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus
lautus, Bacillus lentus, Bacillus licheniformis, Bacillus
megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus
subtilis, or Bacillus thuringiensis polypeptide having enzyme
activity.
[0384] In another aspect, the polypeptide is a Streptococcus
equisimilis, Streptococcus pyogenes, Streptococcus uberis, or
Streptococcus equi subsp. Zooepidemicus polypeptide having enzyme
activity.
[0385] In another aspect, the polypeptide is a Streptomyces
achromogenes, Streptomyces avermitilis, Streptomyces coelicolor,
Streptomyces griseus, or Streptomyces lividans polypeptide having
enzyme activity.
[0386] The polypeptide having enzyme activity may also be a fungal
polypeptide, and more preferably a yeast polypeptide such as a
Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces,
or Yarrowia polypeptide having enzyme activity; or more preferably
a filamentous fungal polypeptide such as an Acremonium, Agaricus,
Alternaria, Aspergillus, Aureobasidium, Botryosphaeria,
Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps,
Cochliobolus, Coprinopsis, Coptotermes, Corynascus, Cryphonectria,
Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella,
Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaeria,
Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora,
Neocallimastix, Neurospora, Paecilomyces, Penicillium,
Phanerochaete, Piromyces, Poitrasia, Pseudoplectania,
Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium,
Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trichoderma,
Trichophaea, Verticillium, Volvariella, or Xylaria polypeptide
having enzyme activity.
[0387] In one aspect, the polypeptide is a Saccharomyces
carlsbergensis, Saccharomyces cerevisiae, Saccharomyces
diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri,
Saccharomyces norbensis, or Saccharomyces oviformis polypeptide
having enzyme activity.
[0388] In another aspect, the polypeptide is an Acremonium
cellulolyticus, Aspergillus aculeatus, Aspergillus awamori,
Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus,
Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,
Chrysosporium keratinophilum, Chrysosporium lucknowense,
Chrysosporium tropicum, Chrysosporium merdarium, Chrysosporium
inops, Chrysosporium pannicola, Chrysosporium queenslandicum,
Chrysosporium zonatum, Fusarium bactridioides, Fusarium cerealis,
Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum,
Fusarium graminum, Fusarium heterosporum, Fusarium negundi,
Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium
sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides,
Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides,
Fusarium venenatum, Humicola grisea, Humicola insolens, Humicola
lanuginosa, Irpex lacteus, Mucor miehei, Myceliophthora
thermophila, Neurospora crassa, Penicillium funiculosum,
Penicillium purpurogenum, Phanerochaete chrysosporium, Thielavia
achromatica, Thielavia albomyces, Thielavia albopilosa, Thielavia
australeinsis, Thielavia fimeti, Thielavia microspora, Thielavia
ovispora, Thielavia peruviana, Thielavia spededonium, Thielavia
setosa, Thielavia subthermophila, Thielavia terrestris, Trichoderma
harzianum, Trichoderma koningii, Trichoderma longibrachiatum,
Trichoderma reesei, Trichoderma viride, or Trichophaea saccata
polypeptide having enzyme activity.
[0389] Chemically modified or protein engineered mutants of
polypeptides having enzyme activity may also be used.
[0390] One or more (e.g., several) components of the enzyme
composition may be a recombinant component, i.e., produced by
cloning of a DNA sequence encoding the single component and
subsequent cell transformed with the DNA sequence and expressed in
a host (see, for example, WO 91/17243 and WO 91/17244). The host
can be a heterologous host (enzyme is foreign to host), but the
host may under certain conditions also be a homologous host (enzyme
is native to host). Monocomponent cellulolytic proteins may also be
prepared by purifying such a protein from a fermentation broth.
[0391] In one aspect, the one or more (e.g., several) cellulolytic
enzymes comprise a commercial cellulolytic enzyme preparation.
Examples of commercial cellulolytic enzyme preparations suitable
for use in the present invention include, for example, CELLIC.RTM.
CTec (Novozymes A/S), CELLIC.RTM. CTec2 (Novozymes A/S),
CELLIC.RTM. CTec3 (Novozymes A/S), CELLUCLAST.TM. (Novozymes A/S),
NOVOZYM.TM. 188 (Novozymes A/S), SPEZYME.TM. CP (Genencor Int.),
ACCELLERASE.TM. TRIO (DuPont), FILTRASE.RTM. NL (DSM);
METHAPLUS.RTM. S/L 100 (DSM), ROHAMENT.TM. 7069 W (Rohm GmbH), or
ALTERNAFUEL.RTM. CMAX3.TM. (Dyadic International, Inc.). The
cellulolytic enzyme preparation is added in an amount effective
from about 0.001 to about 5.0 wt. % of solids, e.g., about 0.025 to
about 4.0 wt. % of solids or about 0.005 to about 2.0 wt. % of
solids.
[0392] Examples of bacterial endoglucanases that can be used in the
processes of the present invention, include, but are not limited
to, Acidothermus cellulolyticus endoglucanase (WO 91/05039; WO
93/15186; U.S. Pat. No. 5,275,944; WO 96/02551; U.S. Pat. No.
5,536,655; WO 00/70031; WO 05/093050), Erwinia carotovara
endoglucanase (Saarilahti et al., 1990, Gene 90: 9-14),
Thermobifida fusca endoglucanase III (WO 05/093050), and
Thermobifida fusca endoglucanase V (WO 05/093050).
[0393] Examples of fungal endoglucanases that can be used in the
present invention, include, but are not limited to, Trichoderma
reesei endoglucanase I (Penttila et al., 1986, Gene 45: 253-263,
Trichoderma reesei Cel7B endoglucanase I (GenBank:M15665),
Trichoderma reesei endoglucanase II (Saloheimo et al., 1988, Gene
63:11-22), Trichoderma reesei Cel5A endoglucanase II
(GenBank:M19373), Trichoderma reesei endoglucanase III (Okada et
al., 1988, Appl. Environ. Microbiol. 64: 555-563,
GenBank:AB003694), Trichoderma reesei endoglucanase V (Saloheimo et
al., 1994, Molecular Microbiology 13: 219-228, GenBank:Z33381),
Aspergillus aculeatus endoglucanase (Ooi et al., 1990, Nucleic
Acids Research 18: 5884), Aspergillus kawachii endoglucanase
(Sakamoto et al., 1995, Current Genetics 27: 435-439), Fusarium
oxysporum endoglucanase (GenBank:L29381), Humicola grisea var.
thermoidea endoglucanase (GenBank:AB003107), Melanocarpus albomyces
endoglucanase (GenBank:MAL515703), Neurospora crassa endoglucanase
(GenBank:XM_324477), Humicola insolens endoglucanase V,
Myceliophthora thermophila CBS 117.65 endoglucanase, Thermoascus
aurantiacus endoglucanase I (GenBank:AF487830), Trichoderma reesei
strain No. VTT-D-80133 endoglucanase (GenBank:M15665), and
Penicillium pinophilum endoglucanase (WO 2012/062220).
[0394] Examples of cellobiohydrolases useful in the present
invention include, but are not limited to, Aspergillus aculeatus
cellobiohydrolase II (WO 2011/059740), Aspergillus fumigatus
cellobiohydrolase I (WO 2013/028928), Aspergillus fumigatus
cellobiohydrolase II (WO 2013/028928), Chaetomium thermophilum
cellobiohydrolase I, Chaetomium thermophilum cellobiohydrolase II,
Humicola insolens cellobiohydrolase I, Myceliophthora thermophila
cellobiohydrolase II (WO 2009/042871), Penicillium occitanis
cellobiohydrolase I (GenBank:AY690482), Talaromyces emersonii
cellobiohydrolase I (GenBank:AF439936), Thielavia hyrcanie
cellobiohydrolase II (WO 2010/141325), Thielavia terrestris
cellobiohydrolase II (CEL6A, WO 2006/074435), Trichoderma reesei
cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, and
Trichophaea saccata cellobiohydrolase II (WO 2010/057086).
[0395] Examples of beta-glucosidases useful in the present
invention include, but are not limited to, beta-glucosidases from
Aspergillus aculeatus (Kawaguchi et al., 1996, Gene 173: 287-288),
Aspergillus fumigatus (WO 2005/047499), Aspergillus niger (Dan et
al., 2000, J. Biol. Chem. 275: 4973-4980), Aspergillus oryzae (WO
02/095014), Penicillium brasilianum IBT 20888 (WO 2007/019442 and
WO 2010/088387), Thielavia terrestris (WO 2011/035029), and
Trichophaea saccata (WO 2007/019442).
[0396] The beta-glucosidase may be a fusion protein. In one aspect,
the beta-glucosidase is an Aspergillus oryzae beta-glucosidase
variant BG fusion protein (WO 2008/057637) or an Aspergillus oryzae
beta-glucosidase fusion protein (WO 2008/057637).
[0397] Other useful endoglucanases, cellobiohydrolases, and
beta-glucosidases are disclosed in numerous Glycosyl Hydrolase
families using the classification according to Henrissat, 1991,
Biochem. J. 280: 309-316, and Henrissat and Bairoch, 1996, Biochem.
J. 316: 695-696.
[0398] Other cellulolytic enzymes that may be used in the present
invention are described in WO 98/13465, WO 98/015619, WO 98/015633,
WO 99/06574, WO 99/10481, WO 99/025847, WO 99/031255, WO
2002/101078, WO 2003/027306, WO 2003/052054, WO 2003/052055, WO
2003/052056, WO 2003/052057, WO 2003/052118, WO 2004/016760, WO
2004/043980, WO 2004/048592, WO 2005/001065, WO 2005/028636, WO
2005/093050, WO 2005/093073, WO 2006/074005, WO 2006/117432, WO
2007/071818, WO 2007/071820, WO 2008/008070, WO 2008/008793, U.S.
Pat. No. 5,457,046, U.S. Pat. No. 5,648,263, and U.S. Pat. No.
5,686,593.
[0399] In the processes of the present invention, any GH61
polypeptide having cellulolytic enhancing activity can be used as a
component of the enzyme composition.
[0400] Examples of GH61 polypeptides useful in the processes of the
present invention include, but are not limited to, GH61
polypeptides from Thielavia terrestris (WO 2005/074647, WO
2008/148131, and WO 2011/035027), Thermoascus aurantiacus (WO
2005/074656 and WO 2010/065830), Trichoderma reesei (WO 2007/089290
and WO 2012/149344), Myceliophthora thermophila (WO 2009/085935, WO
2009/085859, WO 2009/085864, WO 2009/085868, and WO 2009/033071),
Aspergillus fumigatus (WO 2010/138754), Penicillium pinophilum (WO
2011/005867), Thermoascus sp. (WO 2011/039319), Penicillium sp.
(emersonii) (WO 2011/041397 and WO 2012/000892), Thermoascus
crustaceous (WO 2011/041504), Aspergillus aculeatus (WO
2012/125925), Thermomyces lanuginosus (WO 2012/113340, WO
2012/129699, WO 2012/130964, and WO 2012/129699), Aurantiporus
alborubescens (WO 2012/122477), Trichophaea saccata (WO
2012/122477), Penicillium thomii (WO 2012/122477), Talaromyces
stipitatus (WO 2012/135659), Humicola insolens (WO 2012/146171),
Malbranchea cinnamomea (WO 2012/101206), Talaromyces leycettanus
(WO 2012/101206), and Chaetomium thermophilum (WO 2012/101206), and
Talaromyces thermophilus (WO 2012/129697 and WO 2012/130950).
[0401] In one aspect, the GH61 polypeptide having cellulolytic
enhancing activity is used in the presence of a soluble activating
divalent metal cation according to WO 2008/151043, e.g., manganese
or copper.
[0402] In another aspect, the GH61 polypeptide having cellulolytic
enhancing activity is used in the presence of a dioxy compound, a
bicylic compound, a heterocyclic compound, a nitrogen-containing
compound, a quinone compound, a sulfur-containing compound, or a
liquor obtained from a pretreated cellulosic material such as
pretreated corn stover (WO 2012/021394, WO 2012/021395, WO
2012/021396, WO 2012/021399, WO 2012/021400, WO 2012/021401, WO
2012/021408, and WO 2012/021410).
[0403] The dioxy compound may include any suitable compound
containing two or more oxygen atoms. In some aspects, the dioxy
compounds contain a substituted aryl moiety as described herein.
The dioxy compounds may comprise one or more (e.g., several)
hydroxyl and/or hydroxyl derivatives, but also include substituted
aryl moieties lacking hydroxyl and hydroxyl derivatives.
Non-limiting examples of the dioxy compounds include pyrocatechol
or catechol; caffeic acid; 3,4-dihydroxybenzoic acid;
4-tert-butyl-5-methoxy-1,2-benzenediol; pyrogallol; gallic acid;
methyl-3,4,5-trihydroxybenzoate; 2,3,4-trihydroxybenzophenone;
2,6-dimethoxyphenol; sinapinic acid; 3,5-dihydroxybenzoic acid;
4-chloro-1,2-benzenediol; 4-nitro-1,2-benzenediol; tannic acid;
ethyl gallate; methyl glycolate; dihydroxyfumaric acid;
2-butyne-1,4-diol; (croconic acid; 1,3-propanediol; tartaric acid;
2,4-pentanediol; 3-ethyoxy-1,2-propanediol;
2,4,4'-trihydroxybenzophenone; cis-2-butene-1,4-diol;
3,4-dihydroxy-3-cyclobutene-1,2-dione; dihydroxyacetone; acrolein
acetal; methyl-4-hydroxybenzoate; 4-hydroxybenzoic acid; and
methyl-3,5-dimethoxy-4-hydroxybenzoate; or a salt or solvate
thereof.
[0404] The bicyclic compound may include any suitable substituted
fused ring system as described herein. The compounds may comprise
one or more (e.g., several) additional rings, and are not limited
to a specific number of rings unless otherwise stated. In one
aspect, the bicyclic compound is a flavonoid. In another aspect,
the bicyclic compound is an optionally substituted isoflavonoid. In
another aspect, the bicyclic compound is an optionally substituted
flavylium ion, such as an optionally substituted anthocyanidin or
optionally substituted anthocyanin, or derivative thereof.
Non-limiting examples of the bicyclic compounds include
epicatechin; quercetin; myricetin; taxifolin; kaempferol; morin;
acacetin; naringenin; isorhamnetin; apigenin; cyanidin; cyanin;
kuromanin; keracyanin; or a salt or solvate thereof.
[0405] The heterocyclic compound may be any suitable compound, such
as an optionally substituted aromatic or non-aromatic ring
comprising a heteroatom, as described herein. In one aspect, the
heterocyclic is a compound comprising an optionally substituted
heterocycloalkyl moiety or an optionally substituted heteroaryl
moiety. In another aspect, the optionally substituted
heterocycloalkyl moiety or optionally substituted heteroaryl moiety
is an optionally substituted 5-membered heterocycloalkyl or an
optionally substituted 5-membered heteroaryl moiety. In another
aspect, the optionally substituted heterocycloalkyl or optionally
substituted heteroaryl moiety is an optionally substituted moiety
selected from pyrazolyl, furanyl, imidazolyl, isoxazolyl,
oxadiazolyl, oxazolyl, pyrrolyl, pyridyl, pyrimidyl, pyridazinyl,
thiazolyl, triazolyl, thienyl, dihydrothieno-pyrazolyl,
thianaphthenyl, carbazolyl, benzimidazolyl, benzothienyl,
benzofuranyl, indolyl, quinolinyl, benzotriazolyl, benzothiazolyl,
benzooxazolyl, benzimidazolyl, isoquinolinyl, isoindolyl,
acridinyl, benzoisazolyl, dimethylhydantoin, pyrazinyl,
tetrahydrofuranyl, pyrrolinyl, pyrrolidinyl, morpholinyl, indolyl,
diazepinyl, azepinyl, thiepinyl, piperidinyl, and oxepinyl. In
another aspect, the optionally substituted heterocycloalkyl moiety
or optionally substituted heteroaryl moiety is an optionally
substituted furanyl. Non-limiting examples of the heterocyclic
compounds include
(1,2-dihydroxyethyl)-3,4-dihydroxyfuran-2(5H)-one;
4-hydroxy-5-methyl-3-furanone; 5-hydroxy-2(5H)-furanone;
[1,2-dihydroxyethyl]furan-2,3,4(5H)-trione;
.alpha.-hydroxy-.gamma.-butyrolactone; ribonic .gamma.-lactone;
aldohexuronicaldohexuronic acid .gamma.-lactone; gluconic acid
.delta.-lactone; 4-hydroxycoumarin; dihydrobenzofuran;
5-(hydroxymethyl)furfural; furoin; 2(5H)-furanone;
5,6-dihydro-2H-pyran-2-one; and
5,6-dihydro-4-hydroxy-6-methyl-2H-pyran-2-one; or a salt or solvate
thereof.
[0406] The nitrogen-containing compound may be any suitable
compound with one or more nitrogen atoms. In one aspect, the
nitrogen-containing compound comprises an amine, imine,
hydroxylamine, or nitroxide moiety. Non-limiting examples of the
nitrogen-containing compounds include acetone oxime; violuric acid;
pyridine-2-aldoxime; 2-aminophenol; 1,2-benzenediamine;
2,2,6,6-tetramethyl-1-piperidinyloxy; 5,6,7,8-tetrahydrobiopterin;
6,7-dimethyl-5,6,7,8-tetrahydropterine; and maleamic acid; or a
salt or solvate thereof.
[0407] The quinone compound may be any suitable compound comprising
a quinone moiety as described herein. Non-limiting examples of the
quinone compounds include 1,4-benzoquinone; 1,4-naphthoquinone;
2-hydroxy-1,4-naphthoquinone;
2,3-dimethoxy-5-methyl-1,4-benzoquinone or coenzyme Q.sub.0;
2,3,5,6-tetramethyl-1,4-benzoquinone or duroquinone;
1,4-dihydroxyanthraquinone; 3-hydroxy-1-methyl-5,6-indolinedione or
adrenochrome; 4-tert-butyl-5-methoxy-1,2-benzoquinone;
pyrroloquinoline quinone; or a salt or solvate thereof.
[0408] The sulfur-containing compound may be any suitable compound
comprising one or more sulfur atoms. In one aspect, the
sulfur-containing comprises a moiety selected from thionyl,
thioether, sulfinyl, sulfonyl, sulfamide, sulfonamide, sulfonic
acid, and sulfonic ester. Non-limiting examples of the
sulfur-containing compounds include ethanethiol; 2-propanethiol;
2-propene-1-thiol; 2-mercaptoethanesulfonic acid; benzenethiol;
benzene-1,2-dithiol; cysteine; methionine; glutathione; cystine; or
a salt or solvate thereof.
[0409] In one aspect, an effective amount of such a compound
described above is added to cellulosic material at a molar ratio of
the compound to glucosyl units of cellulose of about 10.sup.-6 to
about 10, e.g., about 10.sup.-6 to about 7.5, about 10.sup.-6 to
about 5, about 10.sup.-6 to about 2.5, about 10.sup.-6 to about 1,
about 10.sup.-5 to about 1, about 10.sup.-5 to about 10.sup.-1,
about 10.sup.-4 to about 10.sup.-1, about 10.sup.-3 to about
10.sup.-1, or about 10.sup.-3 to about 10.sup.-2. In another
aspect, an effective amount of such a compound is about 0.1 .mu.M
to about 1 M, e.g., about 0.5 .mu.M to about 0.75 M, about 0.75
.mu.M to about 0.5 M, about 1 .mu.M to about 0.25 M, about 1 .mu.M
to about 0.1 M, about 5 .mu.M to about 50 mM, about 10 .mu.M to
about 25 mM, about 50 .mu.M to about 25 mM, about 10 .mu.M to about
10 mM, about 5 .mu.M to about 5 mM, or about 0.1 mM to about 1
mM.
[0410] The term "liquor" means the solution phase, either aqueous,
organic, or a combination thereof, arising from treatment of a
lignocellulose and/or hemicellulose material in a slurry, or
monosaccharides thereof, e.g., xylose, arabinose, mannose, etc.,
under conditions as described in WO 2012/021401, and the soluble
contents thereof. A liquor for cellulolytic enhancement of a GH61
polypeptide can be produced by treating a lignocellulose or
hemicellulose material (or feedstock) by applying heat and/or
pressure, optionally in the presence of a catalyst, e.g., acid,
optionally in the presence of an organic solvent, and optionally in
combination with physical disruption of the material, and then
separating the solution from the residual solids. Such conditions
determine the degree of cellulolytic enhancement obtainable through
the combination of liquor and a GH61 polypeptide during hydrolysis
of a cellulosic substrate by a cellulolytic enzyme preparation. The
liquor can be separated from the treated material using a method
standard in the art, such as filtration, sedimentation, or
centrifugation.
[0411] In one aspect, an effective amount of the liquor to
cellulose is about 10.sup.-6 to about 10 g per g of cellulose,
e.g., about 10.sup.-6 to about 7.5 g, about 10.sup.-6 to about 5 g,
about 10.sup.-6 to about 2.5 g, about 10.sup.-6 to about 1 g, about
10.sup.-5 to about 1 g, about 10.sup.-5 to about 10.sup.-1 g, about
10.sup.-4 to about 10.sup.-1 g, about 10.sup.-3 to about 10.sup.-1
g, or about 10.sup.-3 to about 10.sup.-2 g per g of cellulose.
[0412] In one aspect, the one or more (e.g., several)
hemicellulolytic enzymes comprise a commercial hemicellulolytic
enzyme preparation. Examples of commercial hemicellulolytic enzyme
preparations suitable for use in the present invention include, for
example, SHEARZYME.TM. (Novozymes A/S), CELLIC.RTM. HTec (Novozymes
A/S), CELLIC.RTM. HTec2 (Novozymes A/S), CELLIC.RTM. HTec3
(Novozymes A/S), VISCOZYME.RTM. (Novozymes A/S), ULTRAFLO.RTM.
(Novozymes A/S), PULPZYME.RTM. HC (Novozymes A/S), MULTIFECT.RTM.
Xylanase (Genencor), ACCELLERASE.RTM. XY (Genencor),
ACCELLERASE.RTM. XC (Genencor), ECOPULP.RTM. TX-200A (AB Enzymes),
HSP 6000 Xylanase (DSM), DEPOL.TM. 333P (Biocatalysts Limit, Wales,
UK), DEPOL.TM. 740L. (Biocatalysts Limit, Wales, UK), and DEPOL.TM.
762P (Biocatalysts Limit, Wales, UK), ALTERNA FUEL 100P (Dyadic),
and ALTERNA FUEL 200P (Dyadic).
[0413] Examples of xylanases useful in the processes of the present
invention include, but are not limited to, xylanases from
Aspergillus aculeatus (GeneSeqP:AAR63790; WO 94/21785), Aspergillus
fumigatus (WO 2006/078256), Penicillium pinophilum (WO
2011/041405), Penicillium sp. (WO 2010/126772), Thermomyces
lanuginosus (GeneSeqP:BAA22485), Talaromyces thermophilus
(GeneSeqP:BAA22834), Thielavia terrestris NRRL 8126 (WO
2009/079210), and Trichophaea saccata (WO 2011/057083).
[0414] Examples of beta-xylosidases useful in the processes of the
present invention include, but are not limited to, beta-xylosidases
from Neurospora crassa (SwissProt:Q7SOW4), Trichoderma reesei
(UniProtKB/TrEMBL:Q92458), Talaromyces emersonii
(SwissProt:Q8X212), and Talaromyces thermophilus
(GeneSeqP:BAA22816). Examples of acetylxylan esterases useful in
the processes of the present invention include, but are not limited
to, acetylxylan esterases from Aspergillus aculeatus (WO
2010/108918), Chaetomium globosum (UniProt:Q2GWX4), Chaetomium
gracile (GeneSeqP:AAB82124), Humicola insolens DSM 1800 (WO
2009/073709), Hypocrea jecorina (WO 2005/001036), Myceliophtera
thermophila (WO 2010/014880), Neurospora crassa (UniProt:q7s259),
Phaeosphaeria nodorum (UniProt:Q0UHJ1), and Thielavia terrestris
NRRL 8126 (WO 2009/042846).
[0415] Examples of feruloyl esterases (ferulic acid esterases)
useful in the processes of the present invention include, but are
not limited to, feruloyl esterases form Humicola insolens DSM 1800
(WO 2009/076122), Neosartorya fischeri (UniProt:A1D9T4), Neurospora
crassa (UniProt:Q9HGR3), Penicillium aurantiogriseum (WO
2009/127729), and Thielavia terrestris (WO 2010/053838 and WO
2010/065448).
[0416] Examples of arabinofuranosidases useful in the processes of
the present invention include, but are not limited to,
arabinofuranosidases from Aspergillus niger (GeneSeqP:AAR94170),
Humicola insolens DSM 1800 (WO 2006/114094 and WO 2009/073383), and
M. giganteus (WO 2006/114094).
[0417] Examples of alpha-glucuronidases useful in the processes of
the present invention include, but are not limited to,
alpha-glucuronidases from Aspergillus clavatus (UniProt:alcc12),
Aspergillus fumigatus (SwissProt:Q4WW45), Aspergillus niger
(UniProt:Q96WX9), Aspergillus terreus (SwissProt:Q0CJP9), Humicola
insolens (WO 2010/014706), Penicillium aurantiogriseum (WO
2009/068565), Talaromyces emersonii (UniProt:Q8X211), and
Trichoderma reesei (UniProt:Q99024).
[0418] Examples of oxidoreductases useful in the processes of the
present invention include, but are not limited to, Aspergillus
fumigatus catalase, Aspergillus lentilus catalase, Aspergillus
niger catalase, Aspergillus oryzae catalase, Humicola insolens
catalase, Neurospora crassa catalase, Penicillium emersonii
catalase, Scytalidium thermophilum catalase, Talaromyces stipitatus
catalase, Thermoascus aurantiacus catalase, Coprinus cinereus
laccase, Myceliophthora thermophila laccase, Polyporus pinsitus
laccase, Pycnoporus cinnabarinus laccase, Rhizoctonia solani
laccase, Streptomyces coelicolor laccase, Coprinus cinereus
peroxidase, Soy peroxidase, and Royal palm peroxidase.
[0419] The polypeptides having enzyme activity used in the
processes of the present invention may be produced by fermentation
of the above-noted microbial strains on a nutrient medium
containing suitable carbon and nitrogen sources and inorganic
salts, using procedures known in the art (see, e.g., Bennett, J. W.
and LaSure, L. (eds.), More Gene Manipulations in Fungi, Academic
Press, C A, 1991). Suitable media are available from commercial
suppliers or may be prepared according to published compositions
(e.g., in catalogues of the American Type Culture Collection).
Temperature ranges and other conditions suitable for growth and
enzyme production are known in the art (see, e.g., Bailey, J. E.,
and Ollis, D. F., Biochemical Engineering Fundamentals, McGraw-Hill
Book Company, N Y, 1986).
[0420] The fermentation can be any method of cultivation of a cell
resulting in the expression or isolation of an enzyme or protein.
Fermentation may, therefore, be understood as comprising shake
flask cultivation, or small- or large-scale fermentation (including
continuous, batch, fed-batch, or solid state fermentations) in
laboratory or industrial fermentors performed in a suitable medium
and under conditions allowing the enzyme to be expressed or
isolated. The resulting enzymes produced by the methods described
above may be recovered from the fermentation medium and purified by
conventional procedures.
[0421] Fermentation. The fermentable sugars obtained from the
hydrolyzed cellulosic material can be fermented by one or more
(e.g., several) fermenting microorganisms capable of fermenting the
sugars directly or indirectly into a desired fermentation product.
"Fermentation" or "fermentation process" refers to any fermentation
process or any process comprising a fermentation step. Fermentation
processes also include fermentation processes used in the
consumable alcohol industry (e.g., beer and wine), dairy industry
(e.g., fermented dairy products), leather industry, and tobacco
industry. The fermentation conditions depend on the desired
fermentation product and fermenting organism and can easily be
determined by one skilled in the art.
[0422] In the fermentation step, sugars, released from the
cellulosic material as a result of the pretreatment and enzymatic
hydrolysis steps, are fermented to a product, e.g., ethanol, by a
fermenting organism, such as yeast. Hydrolysis (saccharification)
and fermentation can be separate or simultaneous.
[0423] Any suitable hydrolyzed cellulosic material can be used in
the fermentation step in practicing the present invention. The
material is generally selected based on economics, i.e., costs per
equivalent sugar potential, and recalcitrance to enzymatic
conversion.
[0424] The term "fermentation medium" is understood herein to refer
to a medium before the fermenting microorganism(s) is(are) added,
such as, a medium resulting from a saccharification process, as
well as a medium used in a simultaneous saccharification and
fermentation process (SSF).
[0425] "Fermenting microorganism" refers to any microorganism,
including bacterial and fungal organisms, suitable for use in a
desired fermentation process to produce a fermentation product. The
fermenting organism can be hexose and/or pentose fermenting
organisms, or a combination thereof. Both hexose and pentose
fermenting organisms are well known in the art. Suitable fermenting
microorganisms are able to ferment, i.e., convert, sugars, such as
glucose, xylose, xylulose, arabinose, maltose, mannose, galactose,
and/or oligosaccharides, directly or indirectly into the desired
fermentation product. Examples of bacterial and fungal fermenting
organisms producing ethanol are described by Lin et al., 2006,
Appl. Microbiol. Biotechnol. 69: 627-642.
[0426] Examples of fermenting microorganisms that can ferment
hexose sugars include bacterial and fungal organisms, such as
yeast. Yeast include strains of Candida, Kluyveromyces, and
Saccharomyces, e.g., Candida sonorensis, Kluyveromyces marxianus,
and Saccharomyces cerevisiae.
[0427] Examples of fermenting organisms that can ferment pentose
sugars in their native state include bacterial and fungal
organisms, such as some yeast. Xylose fermenting yeast include
strains of Candida, preferably C. sheatae or C. sonorensis; and
strains of Pichia, e.g., P. stipitis, such as P. stipitis CBS 5773.
Pentose fermenting yeast include strains of Pachysolen, preferably
P. tannophilus. Organisms not capable of fermenting pentose sugars,
such as xylose and arabinose, may be genetically modified to do so
by methods known in the art. Examples of bacteria that can
efficiently ferment hexose and pentose to ethanol include, for
example, Bacillus coagulans, Clostridium acetobutylicum,
Clostridium thermocellum, Clostridium phytofermentans, Geobacillus
sp., Thermoanaerobacter saccharolyticum, and Zymomonas mobilis
(Philippidis, G. P., 1996, Cellulose bioconversion technology, in
Handbook on Bioethanol: Production and Utilization, Wyman, C. E.,
ed., Taylor & Francis, Washington, D.C., 179-212).
[0428] Other fermenting organisms include strains of Bacillus, such
as Bacillus coagulans; Candida, such as C. sonorensis, C.
methanosorbosa, C. diddensiae, C. parapsilosis, C. naedodendra, C.
blankii, C. entomophilia, C. brassicae, C. pseudotropicalis, C.
boidinii, C. utilis, and C. scehatae; Clostridium, such as C.
acetobutylicum, C. thermocellum, and C. phytofermentans; E. coli,
especially E. coli strains that have been genetically modified to
improve the yield of ethanol; Geobacillus sp.; Hansenula, such as
Hansenula anomala; Klebsiella, such as K. oxytoca; Kluyveromyces,
such as K. marxianus, K. lactis, K. thermotolerans, and K.
fragilis; Schizosaccharomyces, such as S. pombe;
Thermoanaerobacter, such as Thermoanaerobacter saccharolyticum; and
Zymomonas, such as Zymomonas mobilis.
[0429] Commercially available yeast suitable for ethanol production
include, e.g., BIOFERM.TM. AFT and XR (NABC--North American
Bioproducts Corporation, GA, USA), ETHANOL RED.TM. yeast
(Fermentis/Lesaffre, USA), FALI.TM. (Fleischmann's Yeast, USA),
FERMIOL.TM. (DSM Specialties), GERT STRAND.TM. (Gert Strand AB,
Sweden), and SUPERSTART.TM. and THERMOSACC.TM. fresh yeast (Ethanol
Technology, Wis., USA).
[0430] In an aspect, the fermenting microorganism has been
genetically modified to provide the ability to ferment pentose
sugars, such as xylose utilizing, arabinose utilizing, and xylose
and arabinose co-utilizing microorganisms.
[0431] The cloning of heterologous genes into various fermenting
microorganisms has led to the construction of organisms capable of
converting hexoses and pentoses to ethanol (co-fermentation) (Chen
and Ho, 1993, Appl. Biochem. Biotechnol. 39-40: 135-147; Ho et al.,
1998, Appl. Environ. Microbiol. 64: 1852-1859; Kotter and Ciriacy,
1993, Appl. Microbiol. Biotechnol. 38: 776-783; Walfridsson et al.,
1995, Appl. Environ. Microbiol. 61: 4184-4190; Kuyper et al., 2004,
FEMS Yeast Research 4: 655-664; Beall et al., 1991, Biotech.
Bioeng. 38: 296-303; Ingram et al., 1998, Biotechnol. Bioeng. 58:
204-214; Zhang et al., 1995, Science 267: 240-243; Deanda et al.,
1996, Appl. Environ. Microbiol. 62: 4465-4470; WO 03/062430).
[0432] In one aspect, the fermenting organism comprises a
polynucleotide encoding a variant of the present invention.
[0433] In another aspect, the fermenting organism comprises one or
more polynucleotides encoding one or more cellulolytic enzymes,
hemicellulolytic enzymes, and accessory enzymes described
herein.
[0434] It is well known in the art that the organisms described
above can also be used to produce other substances, as described
herein.
[0435] The fermenting microorganism is typically added to the
degraded cellulosic material or hydrolysate and the fermentation is
performed for about 8 to about 96 hours, e.g., about 24 to about 60
hours. The temperature is typically between about 26.degree. C. to
about 60.degree. C., e.g., about 32.degree. C. or 50.degree. C.,
and about pH 3 to about pH 8, e.g., pH 4-5, 6, or 7.
[0436] In one aspect, the yeast and/or another microorganism are
applied to the degraded cellulosic material and the fermentation is
performed for about 12 to about 96 hours, such as typically 24-60
hours. In another aspect, the temperature is preferably between
about 20.degree. C. to about 60.degree. C., e.g., about 25.degree.
C. to about 50.degree. C., about 32.degree. C. to about 50.degree.
C., or about 32.degree. C. to about 50.degree. C., and the pH is
generally from about pH 3 to about pH 7, e.g., about pH 4 to about
pH 7. However, some fermenting organisms, e.g., bacteria, have
higher fermentation temperature optima. Yeast or another
microorganism is preferably applied in amounts of approximately
10.sup.5 to 10.sup.12, preferably from approximately 10.sup.7 to
10.sup.10, especially approximately 2.times.10.sup.8 viable cell
count per ml of fermentation broth. Further guidance in respect of
using yeast for fermentation can be found in, e.g., "The Alcohol
Textbook" (Editors K. Jacques, T. P. Lyons and D. R. Kelsall,
Nottingham University Press, United Kingdom 1999), which is hereby
incorporated by reference.
[0437] A fermentation stimulator can be used in combination with
any of the processes described herein to further improve the
fermentation process, and in particular, the performance of the
fermenting microorganism, such as, rate enhancement and ethanol
yield. A "fermentation stimulator" refers to stimulators for growth
of the fermenting microorganisms, in particular, yeast. Preferred
fermentation stimulators for growth include vitamins and minerals.
Examples of vitamins include multivitamins, biotin, pantothenate,
nicotinic acid, meso-inositol, thiamine, pyridoxine,
para-aminobenzoic acid, folic acid, riboflavin, and Vitamins A, B,
C, D, and E. See, for example, Alfenore et al., Improving ethanol
production and viability of Saccharomyces cerevisiae by a vitamin
feeding strategy during fed-batch process, Springer-Verlag (2002),
which is hereby incorporated by reference. Examples of minerals
include minerals and mineral salts that can supply nutrients
comprising P, K, Mg, S, Ca, Fe, Zn, Mn, and Cu.
[0438] Fermentation products: A fermentation product can be any
substance derived from the fermentation. The fermentation product
can be, without limitation, an alcohol (e.g., arabinitol,
n-butanol, isobutanol, ethanol, glycerol, methanol, ethylene
glycol, 1,3-propanediol [propylene glycol], butanediol, glycerin,
sorbitol, and xylitol); an alkane (e.g., pentane, hexane, heptane,
octane, nonane, decane, undecane, and dodecane), a cycloalkane
(e.g., cyclopentane, cyclohexane, cycloheptane, and cyclooctane),
an alkene (e.g. pentene, hexene, heptene, and octene); an amino
acid (e.g., aspartic acid, glutamic acid, glycine, lysine, serine,
and threonine); a gas (e.g., methane, hydrogen (H.sub.2), carbon
dioxide (CO.sub.2), and carbon monoxide (CO)); isoprene; a ketone
(e.g., acetone); an organic acid (e.g., acetic acid, acetonic acid,
adipic acid, ascorbic acid, citric acid, 2,5-diketo-D-gluconic
acid, formic acid, fumaric acid, glucaric acid, gluconic acid,
glucuronic acid, glutaric acid, 3-hydroxypropionic acid, itaconic
acid, lactic acid, malic acid, malonic acid, oxalic acid,
oxaloacetic acid, propionic acid, succinic acid, and xylonic acid);
and polyketide.
[0439] In one aspect, the fermentation product is an alcohol. The
term "alcohol" encompasses a substance that contains one or more
hydroxyl moieties. The alcohol can be, but is not limited to,
n-butanol, isobutanol, ethanol, methanol, arabinitol, butanediol,
ethylene glycol, glycerin, glycerol, 1,3-propanediol, sorbitol,
xylitol. See, for example, Gong et al., 1999, Ethanol production
from renewable resources, in Advances in Biochemical
Engineering/Biotechnology, Scheper, T., ed., Springer-Verlag Berlin
Heidelberg, Germany, 65: 207-241; Silveira and Jonas, 2002, Appl.
Microbiol. Biotechnol. 59: 400-408; Nigam and Singh, 1995, Process
Biochemistry 30(2): 117-124; Ezeji et al., 2003, World Journal of
Microbiology and Biotechnology 19(6): 595-603.
[0440] In another aspect, the fermentation product is an alkane.
The alkane may be an unbranched or a branched alkane. The alkane
can be, but is not limited to, pentane, hexane, heptane, octane,
nonane, decane, undecane, or dodecane.
[0441] In another aspect, the fermentation product is a
cycloalkane. The cycloalkane can be, but is not limited to,
cyclopentane, cyclohexane, cycloheptane, or cyclooctane.
[0442] In another aspect, the fermentation product is an alkene.
The alkene may be an unbranched or a branched alkene. The alkene
can be, but is not limited to, pentene, hexene, heptene, or
octene.
[0443] In another aspect, the fermentation product is an amino
acid. The organic acid can be, but is not limited to, aspartic
acid, glutamic acid, glycine, lysine, serine, or threonine. See,
for example, Richard and Margaritis, 2004, Biotechnology and
Bioengineering 87(4): 501-515.
[0444] In another aspect, the fermentation product is a gas. The
gas can be, but is not limited to, methane, H.sub.2, CO.sub.2, or
CO. See, for example, Kataoka et al., 1997, Water Science and
Technology 36(6-7): 41-47; and Gunaseelan, 1997, Biomass and
Bioenergy 13(1-2): 83-114.
[0445] In another aspect, the fermentation product is isoprene.
[0446] In another aspect, the fermentation product is a ketone. The
term "ketone" encompasses a substance that contains one or more
ketone moieties. The ketone can be, but is not limited to,
acetone.
[0447] In another aspect, the fermentation product is an organic
acid. The organic acid can be, but is not limited to, acetic acid,
acetonic acid, adipic acid, ascorbic acid, citric acid,
2,5-diketo-D-gluconic acid, formic acid, fumaric acid, glucaric
acid, gluconic acid, glucuronic acid, glutaric acid,
3-hydroxypropionic acid, itaconic acid, lactic acid, malic acid,
malonic acid, oxalic acid, propionic acid, succinic acid, or
xylonic acid. See, for example, Chen and Lee, 1997, Appl. Biochem.
Biotechnol. 63-65: 435-448.
[0448] In another aspect, the fermentation product is
polyketide.
[0449] Recovery.
[0450] The fermentation product(s) can be optionally recovered from
the fermentation medium using any method known in the art
including, but not limited to, chromatography, electrophoretic
procedures, differential solubility, distillation, or extraction.
For example, alcohol is separated from the fermented cellulosic
material and purified by conventional methods of distillation.
Ethanol with a purity of up to about 96 vol. % can be obtained,
which can be used as, for example, fuel ethanol, drinking ethanol,
i.e., potable neutral spirits, or industrial ethanol.
Plants
[0451] The present invention also relates to isolated plants, e.g.,
a transgenic plant, plant part, or plant cell, comprising a
polynucleotide of the present invention so as to express and
produce a cellobiohydrolase variant in recoverable quantities. The
variant may be recovered from the plant or plant part.
Alternatively, the plant or plant part containing the variant may
be used as such for improving the quality of a food or feed, e.g.,
improving nutritional value, palatability, and rheological
properties, or to destroy an antinutritive factor.
[0452] The transgenic plant can be dicotyledonous (a dicot) or
monocotyledonous (a monocot). Examples of monocot plants are
grasses, such as meadow grass (blue grass, Poa), forage grass such
as Festuca, Lolium, temperate grass, such as Agrostis, and cereals,
e.g., wheat, oats, rye, barley, rice, sorghum, and maize
(corn).
[0453] Examples of dicot plants are tobacco, legumes, such as
lupins, potato, sugar beet, pea, bean and soybean, and cruciferous
plants (family Brassicaceae), such as cauliflower, rape seed, and
the closely related model organism Arabidopsis thaliana.
[0454] Examples of plant parts are stem, callus, leaves, root,
fruits, seeds, and tubers as well as the individual tissues
comprising these parts, e.g., epidermis, mesophyll, parenchyme,
vascular tissues, meristems. Specific plant cell compartments, such
as chloroplasts, apoplasts, mitochondria, vacuoles, peroxisomes and
cytoplasm are also considered to be a plant part. Furthermore, any
plant cell, whatever the tissue origin, is considered to be a plant
part. Likewise, plant parts such as specific tissues and cells
isolated to facilitate the utilization of the invention are also
considered plant parts, e.g., embryos, endosperms, aleurone and
seed coats.
[0455] Also included within the scope of the present invention are
the progeny of such plants, plant parts, and plant cells.
[0456] The transgenic plant or plant cell expressing a variant may
be constructed in accordance with methods known in the art. In
short, the plant or plant cell is constructed by incorporating one
or more expression constructs encoding a variant into the plant
host genome or chloroplast genome and propagating the resulting
modified plant or plant cell into a transgenic plant or plant
cell.
[0457] The expression construct is conveniently a nucleic acid
construct that comprises a polynucleotide encoding a variant
operably linked with appropriate regulatory sequences required for
expression of the polynucleotide in the plant or plant part of
choice. Furthermore, the expression construct may comprise a
selectable marker useful for identifying plant cells into which the
expression construct has been integrated and DNA sequences
necessary for introduction of the construct into the plant in
question (the latter depends on the DNA introduction method to be
used).
[0458] The choice of regulatory sequences, such as promoter and
terminator sequences and optionally signal or transit sequences, is
determined, for example, on the basis of when, where, and how the
variant is desired to be expressed (Sticklen, 2008, Nature Reviews
9: 433-443). For instance, the expression of the gene encoding a
variant may be constitutive or inducible, or may be developmental,
stage or tissue specific, and the gene product may be targeted to a
specific tissue or plant part such as seeds or leaves. Regulatory
sequences are, for example, described by Tague et al., 1988, Plant
Physiology 86: 506.
[0459] For constitutive expression, the 35S-CaMV, the maize
ubiquitin 1, or the rice actin 1 promoter may be used (Franck et
al., 1980, Cell 21: 285-294; Christensen et al., 1992, Plant Mol.
Biol. 18: 675-689; Zhang et al., 1991, Plant Cell 3: 1155-1165).
Organ-specific promoters may be, for example, a promoter from
storage sink tissues such as seeds, potato tubers, and fruits
(Edwards and Coruzzi, 1990, Ann. Rev. Genet. 24: 275-303), or from
metabolic sink tissues such as meristems (Ito et al., 1994, Plant
Mol. Biol. 24: 863-878), a seed specific promoter such as the
glutelin, prolamin, globulin, or albumin promoter from rice (Wu et
al., 1998, Plant Cell Physiol. 39: 885-889), a Vicia faba promoter
from the legumin B4 and the unknown seed protein gene from Vicia
faba (Conrad et al., 1998, J. Plant Physiol. 152: 708-711), a
promoter from a seed oil body protein (Chen et al., 1998, Plant
Cell Physiol. 39: 935-941), the storage protein napA promoter from
Brassica napus, or any other seed specific promoter known in the
art, e.g., as described in WO 91/14772. Furthermore, the promoter
may be a leaf specific promoter such as the rbcs promoter from rice
or tomato (Kyozuka et al., 1993, Plant Physiol. 102: 991-1000), the
chlorella virus adenine methyltransferase gene promoter (Mitra and
Higgins, 1994, Plant Mol. Biol. 26: 85-93), the aldP gene promoter
from rice (Kagaya et al., 1995, Mol. Gen. Genet. 248: 668-674), or
a wound inducible promoter such as the potato pin2 promoter (Xu et
al., 1993, Plant Mol. Biol. 22: 573-588). Likewise, the promoter
may be induced by abiotic treatments such as temperature, drought,
or alterations in salinity or induced by exogenously applied
substances that activate the promoter, e.g., ethanol, oestrogens,
plant hormones such as ethylene, abscisic acid, and gibberellic
acid, and heavy metals.
[0460] A promoter enhancer element may also be used to achieve
higher expression of a variant in the plant. For instance, the
promoter enhancer element may be an intron that is placed between
the promoter and the polynucleotide encoding a variant. For
instance, Xu et al., 1993, supra, disclose the use of the first
intron of the rice actin 1 gene to enhance expression.
[0461] The selectable marker gene and any other parts of the
expression construct may be chosen from those available in the
art.
[0462] The nucleic acid construct is incorporated into the plant
genome according to conventional techniques known in the art,
including Agrobacterium-mediated transformation, virus-mediated
transformation, microinjection, particle bombardment, biolistic
transformation, and electroporation (Gasser et al., 1990, Science
244: 1293; Potrykus, 1990, Bio/Technology 8: 535; Shimamoto et al.,
1989, Nature 338: 274).
[0463] Agrobacterium tumefaciens-mediated gene transfer is a method
for generating transgenic dicots (for a review, see Hooykas and
Schilperoort, 1992, Plant Mol. Biol. 19: 15-38) and for
transforming monocots, although other transformation methods may be
used for these plants. A method for generating transgenic monocots
is particle bombardment (microscopic gold or tungsten particles
coated with the transforming DNA) of embryonic calli or developing
embryos (Christou, 1992, Plant J. 2: 275-281; Shimamoto, 1994,
Curr. Opin. Biotechnol. 5: 158-162; Vasil et al., 1992,
Bio/Technology 10: 667-674). An alternative method for
transformation of monocots is based on protoplast transformation as
described by Omirulleh et al., 1993, Plant Mol. Biol. 21: 415-428.
Additional transformation methods include those described in U.S.
Pat. Nos. 6,395,966 and 7,151,204 (both of which are herein
incorporated by reference in their entirety).
[0464] Following transformation, the transformants having
incorporated the expression construct are selected and regenerated
into whole plants according to methods well known in the art. Often
the transformation procedure is designed for the selective
elimination of selection genes either during regeneration or in the
following generations by using, for example, co-transformation with
two separate T-DNA constructs or site specific excision of the
selection gene by a specific recombinase.
[0465] In addition to direct transformation of a particular plant
genotype with a construct of the present invention, transgenic
plants may be made by crossing a plant having the construct to a
second plant lacking the construct. For example, a construct
encoding a variant can be introduced into a particular plant
variety by crossing, without the need for ever directly
transforming a plant of that given variety. Therefore, the present
invention encompasses not only a plant directly regenerated from
cells which have been transformed in accordance with the present
invention, but also the progeny of such plants. As used herein,
progeny may refer to the offspring of any generation of a parent
plant prepared in accordance with the present invention. Such
progeny may include a DNA construct prepared in accordance with the
present invention. Crossing results in the introduction of a
transgene into a plant line by cross pollinating a starting line
with a donor plant line. Non-limiting examples of such steps are
described in U.S. Pat. No. 7,151,204.
[0466] Plants may be generated through a process of backcross
conversion. For example, plants include plants referred to as a
backcross converted genotype, line, inbred, or hybrid.
[0467] Genetic markers may be used to assist in the introgression
of one or more transgenes of the invention from one genetic
background into another. Marker assisted selection offers
advantages relative to conventional breeding in that it can be used
to avoid errors caused by phenotypic variations. Further, genetic
markers may provide data regarding the relative degree of elite
germplasm in the individual progeny of a particular cross. For
example, when a plant with a desired trait which otherwise has a
non-agronomically desirable genetic background is crossed to an
elite parent, genetic markers may be used to select progeny which
not only possess the trait of interest, but also have a relatively
large proportion of the desired germplasm. In this way, the number
of generations required to introgress one or more traits into a
particular genetic background is minimized.
[0468] The present invention also relates to methods of producing a
variant of the present invention comprising: (a) cultivating a
transgenic plant or a plant cell comprising a polynucleotide
encoding the variant under conditions conducive for production of
the variant; and optionally (b) recovering the variant.
[0469] The invention may be further described by the following
numbered paragraphs:
[1] A cellobiohydrolase variant, comprising an alteration at one or
more (e.g., several) positions corresponding to positions 4, 44,
45, 72, 265, 266, 391, 393 and 394 of SEQ ID NO: 4, wherein the
variant has cellobiohydrolase activity, and wherein the variant has
at least 60%, e.g., at least 65%, at least 70%, at least 75%, at
least 80%, at least 81%, at least 82%, at least 83%, at least 84%,
at least 85%, at least 86%, at least 87%, at least 88%, at least
89%, at least 90%, at least 95%, at least 96%, at least 97%, at
least 98%, or at least 99%, but less than 100%, sequence identity
to the mature polypeptide of a parent cellobiohydrolase. [2] The
variant of paragraph [1], wherein the alteration is a substitution.
[3] The variant of paragraph [1] or [2], wherein the parent
cellobiohydrolase is selected from the group consisting of: (a) a
polypeptide having at least 60% sequence identity to the mature
polypeptide of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO:
10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ
ID NO: 20, SEQ ID NO: 22, or SEQ ID NO: 52; (b) a polypeptide
encoded by a polynucleotide that hybridizes under low stringency
conditions with (i) the mature polypeptide coding sequence of SEQ
ID NO: 1, SEQ ID NO: 5, ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ
ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:
21, or SEQ ID NO: 51, (ii) the cDNA sequence thereof, or (iii) the
full-length complement of (i) or (ii); (c) a polypeptide encoded by
a polynucleotide having at least 60% identity to the mature
polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID
NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15,
SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 51 or the
cDNA sequence thereof; and (d) a fragment of the mature polypeptide
of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID
NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20,
SEQ ID NO: 22, or SEQ ID NO: 52 which has cellobiohydrolase
activity. [4] The variant of paragraph [3], wherein the parent
cellobiohydrolase has at least 60%, at least 65%, at least 70%, at
least 75%, at least 80%, at least 81%, at least 82%, at least 83%,
at least 84%, at least 85%, at least 86%, at least 87%, at least
88%, at least 89%, at least 90%, at least 95%, at least 96%, at
least 97%, at least 98%, at least 99%, or 100% sequence identity to
the mature polypeptide of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8,
SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID
NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, or SEQ ID NO: 52. [5] The
variant of paragraph [3] or [4], wherein the parent
cellobiohydrolase is encoded by a polynucleotide that hybridizes
under low stringency conditions, medium stringency conditions,
medium-high stringency conditions, high stringency conditions, or
very high stringency conditions with (i) the mature polypeptide
coding sequence of SEQ ID NO: 1, SEQ ID NO: 5, ID NO: 7, SEQ ID NO:
9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ
ID NO: 19, SEQ ID NO: 21, or SEQ ID NO: 51, (ii) the cDNA sequence
thereof, or (iii) the full-length complement of (i) or (ii). [6]
The variant of any of paragraphs [3]-[5], wherein the parent
cellobiohydrolase is encoded by a polynucleotide having at least
60%, at least 65%, at least 70%, at least 75%, at least 80%, at
least 81%, at least 82%, at least 83%, at least 84%, at least 85%,
at least 86%, at least 87%, at least 88%, at least 89%, at least
90%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99%, or 100% sequence identity to the mature polypeptide
coding sequence of SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID
NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17,
SEQ ID NO: 19, SEQ ID NO: 21, or SEQ ID NO: 51, or the cDNA
sequence thereof. [7] The variant of any of paragraphs [3]-[5],
wherein the parent cellobiohydrolase comprises or consists of the
mature polypeptide of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ
ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO:
18, SEQ ID NO: 20, SEQ ID NO: 22, or SEQ ID NO: 52. [8] The variant
of any of paragraphs [3]-[7], wherein the parent cellobiohydrolase
is a fragment of the mature polypeptide of SEQ ID NO: 4, SEQ ID NO:
6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ
ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22 or SEQ ID
NO: 52, wherein the fragment has cellobiohydrolase activity. [9]
The variant of any of paragraphs [1]-[8], wherein the number of
alterations is 1-9, e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9 alterations.
[10] The variant of any of paragraphs [1]-[9], which comprises a
substitution at a position corresponding to position 4 of SEQ ID
NO: 4. [11] The variant of paragraph [10], wherein the substitution
is with Cys. [12] The variant of paragraph [11], wherein the
substitution is a Gly to Cys substitution. [13] The variant of any
of paragraphs [1]-[11], which comprises a substitution at a
position corresponding to position 44 of SEQ ID NO: 4. [14] The
variant of paragraph [13], wherein the substitution is with Ser,
Thr, Ala, Gly, Ile, Met, Lys, or Asn. [15] The variant of paragraph
[14], wherein the substitution is a Val to Ser, Thr, Ala, Gly, Ile,
Met, Lys, or Asn substitution. [16] The variant of any of
paragraphs [1]-[15], which comprises a substitution at a position
corresponding to position 45 of SEQ ID NO: 4. [17] The variant of
paragraph [16], wherein the substitution is with Ser or Asn. [18]
The variant of paragraph [17], wherein the substitution is a Gly to
Ser or Asn substitution. [19] The variant of any of paragraphs
[1]-[18], which comprises a substitution at a position
corresponding to position 72 of SEQ ID NO: 4. [20] The variant of
paragraph [19], wherein the substitution is with Cys. [21] The
variant of paragraph [20], wherein the substitution is a Ala to Cys
substitution. [22] The variant of any of paragraphs [1]-[21], which
comprises a substitution at a position corresponding to position
265 of SEQ ID NO: 4. [23] The variant of paragraph [22], wherein
the substitution is with Gly, Pro, or Ala. [24] The variant of
paragraph [23], wherein the substitution is a Ser to Gly, Pro, or
Ala substitution. [25] The variant of any of paragraphs [1]-[24],
which comprises a substitution at a position corresponding to
position 266 of SEQ ID NO: 4. [26] The variant of paragraph [25],
wherein the substitution is with Tyr. [27] The variant of paragraph
[26], wherein the substitution is a Phe to Tyr substitution. [28]
The variant of any of paragraphs [1]-[27], which comprises a
substitution at a position corresponding to position 391 of SEQ ID
NO: 4. [29] The variant of paragraph [28], wherein the substitution
is with Asp, Trp, Asn or Val. [30] The variant of paragraph [29],
wherein the substitution is a Thr to Asp, Trp, Asn or Val
substitution. [31] The variant of any of paragraphs [1]-[30], which
comprises a substitution at a position corresponding to position
393 of SEQ ID NO: 4. [32] The variant of paragraph [31], wherein
the substitution is with Asp. [33] The variant of paragraph [32],
wherein the substitution is a Ser to Asp substitution. [34] The
variant of any of paragraphs [1]-[33], which comprises a
substitution at a position corresponding to position 394 of SEQ ID
NO: 4. [35] The variant of paragraph [34], wherein the substitution
is with Pro. [36] The variant of paragraph [35], wherein the
substitution is a Ser to Pro substitution. [37] The variant of any
of paragraphs [1]-[36], which comprises or consists of SEQ ID NO:
26 or the mature polypeptide thereof (e.g., amino acids 1-506 of
SEQ ID NO: 26), SEQ ID NO: 28 or the mature polypeptide thereof
(e.g., amino acids 1-506 of SEQ ID NO: 28), SEQ ID NO: 38 or the
mature polypeptide thereof (e.g., amino acids 1-506 of SEQ ID NO:
38), SEQ ID NO: 42 or the mature polypeptide thereof (e.g., amino
acids 1-506 of SEQ ID NO: 42), SEQ ID NO: 46 or the mature
polypeptide thereof (e.g., amino acids 1-506 of SEQ ID NO: 46), SEQ
ID NO: 48 or the mature polypeptide thereof (e.g., amino acids
1-506 of SEQ ID NO: 48), SEQ ID NO: 63 or the mature polypeptide
thereof (e.g., amino acids 1-503 of SEQ ID NO: 63), SEQ ID NO: 67
or the mature polypeptide thereof (e.g., amino acids 1-503 of SEQ
ID NO: 67), SEQ ID NO: 69 or the mature polypeptide thereof (e.g.,
amino acids 1-503 of SEQ ID NO: 69), SEQ ID NO: 71 or the mature
polypeptide thereof (e.g., amino acids 1-503 of SEQ ID NO: 71), SEQ
ID NO: 73 or the mature polypeptide thereof (e.g., amino acids
1-503 of SEQ ID NO: 73), SEQ ID NO: 75 or the mature polypeptide
thereof (e.g., amino acids 1-503 of SEQ ID NO: 75), SEQ ID NO: 77
or the mature polypeptide thereof (e.g., amino acids 1-503 of SEQ
ID NO: 77), SEQ ID NO: 79 or the mature polypeptide thereof (e.g.,
amino acids 1-503 of SEQ ID NO: 79), SEQ ID NO: 81 or the mature
polypeptide thereof (e.g., amino acids 1-503 of SEQ ID NO: 81), SEQ
ID NO: 83 or the mature polypeptide thereof (e.g., amino acids
1-503 of SEQ ID NO: 83), or SEQ ID NO: 85 or the mature polypeptide
thereof (e.g., amino acids 1-503 of SEQ ID NO: 85). [38] The
variant of any of paragraphs [1]-[38], wherein the parent is a
hybrid or chimeric polypeptide in which the carbohydrate binding
domain of the parent is replaced with a different carbohydrate
binding domain. [39] The variant of any of paragraphs [1]-[38],
which is a hybrid or chimeric polypeptide in which the carbohydrate
binding domain of the variant is replaced with a different
carbohydrate binding domain. [40] The variant of any of paragraphs
[1]-[38], wherein the parent is a fusion protein in which a
heterologous carbohydrate binding domain is fused to the parent.
[41] The variant of paragraph [40], wherein the carbohydrate
binding domain is fused to the N-terminus or the C-terminus of the
parent. [42] The variant of paragraph [40] or [41], wherein the
parent comprises or consists of SEQ ID NO: 24 or the mature
polypeptide thereof. [43] The variant of any of paragraphs
[1]-[42], which is a fusion protein in which a heterologous
carbohydrate binding domain is fused to the variant. [44] The
variant of paragraph [43], wherein the carbohydrate binding domain
is fused to the N-terminus or the C-terminus of the variant. [45]
The variant of any of paragraphs [1]-[44], which has an increased
specific performance relative to the parent. [46] A
cellobiohydrolase variant, comprising an alteration at one or more
(e.g., several) positions corresponding to positions 4, 44, 45, 72,
265, 266, 391, 393 and 394 of SEQ ID NO: 4, wherein the variant has
cellobiohydrolase activity, and wherein the variant comprises a
catalytic domain having at least 60%, e.g., at least 65%, at least
70%, at least 75%, at least 80%, at least 81%, at least 82%, at
least 83%, at least 84%, at least 85%, at least 86%, at least 87%,
at least 88%, at least 89%, at least 90%, at least 95%, at least
96%, at least 97%, at least 98%, or at least 99%, but less than
100%, sequence identity to the catalytic domain of a parent
cellobiohydrolase. [47] The variant of paragraph [46], wherein the
alteration is a substitution. [48] The variant of paragraph [47] or
[47], wherein the parent cellobiohydrolase comprises a catalytic
domain having at least 60%, at least 65%, at least 70%, at least
75%, at least 80%, at least 81%, at least 82%, at least 83%, at
least 84%, at least 85%, at least 86%, at least 87%, at least 88%,
at least 89%, at least 90%, at least 95%, at least 96%, at least
97%, at least 98%, at least 99%, or 100% sequence identity to the
amino acids 1 to 437 of SEQ ID NO: 4, amino acids 1 to 429 of SEQ
ID NO: 6, amino acids 1 to 440 of SEQ ID NO: 8, amino acids 1 to
437 of SEQ ID NO: 10, amino acids 1 to 437 of SEQ ID NO: 12, amino
acids 1 to 437 of SEQ ID NO: 14, amino acids 1 to 437 of SEQ ID NO:
16, amino acids 1 to 430 of SEQ ID NO: 18, amino acids 1 to 433 of
SEQ ID NO: 20, amino acids 1 to 438 of SEQ ID NO: 22 or amino acids
1 to 437 of SEQ ID NO: 52. [49] The variant of any of paragraphs
[46]-[48], wherein the parent cellobiohydrolase comprises a
catalytic domain comprising or consisting of amino acids 1 to 437
of SEQ ID NO: 4, amino acids 1 to 429 of SEQ ID NO: 6, amino acids
1 to 440 of SEQ ID NO: 8, amino acids 1 to 437 of SEQ ID NO: 10,
amino acids 1 to 437 of SEQ ID NO: 12, amino acids 1 to 437 of SEQ
ID NO: 14, amino acids 1 to 437 of SEQ ID NO: 16, amino acids 1 to
430 of SEQ ID NO: 18, amino acids 1 to 433 of SEQ ID NO: 20, amino
acids 1 to 438 of SEQ ID NO: 22, or amino acids 1 to 437 of SEQ ID
NO: 52. [50] The variant of any of paragraphs [46]-[49], wherein
the number of alterations is 1-9, e.g., 1, 2, 3, 4, 5, 6, 7, 8, or
9 alterations. [51] The variant of any of paragraphs [46]-[50],
which comprises a substitution at a position corresponding to
position 4 of SEQ ID NO: 4. [52] The variant of paragraph [51],
wherein the substitution is with Cys. [53] The variant of paragraph
[52], wherein the substitution is a Gly to Cys substitution. [54]
The variant of any of paragraphs [46]-[53], which comprises a
substitution at a position corresponding to position 44 of SEQ ID
NO: 4. [55] The variant of paragraph [54], wherein the substitution
is with Ser, Thr, Ala, Gly, Ile, Met, Asn, or Lys. [56] The variant
of paragraph [55], wherein the substitution is a Val to Ser, Thr,
Ala, Gly, Ile, Met, Asn, or Lys substitution. [57] The variant of
any of paragraphs [46]-[56], which comprises a substitution at a
position corresponding to position 45 of SEQ ID NO: 4. [58] The
variant of paragraph [57], wherein the substitution is with Ser or
Asn. [59] The variant of paragraph [58], wherein the substitution
is a Gly to Ser or Asn substitution. [60] The variant of any of
paragraphs [46]-[59], which comprises a substitution at a position
corresponding to position 72 of SEQ ID NO: 4. [61] The variant of
paragraph [60], wherein the substitution is with Cys. [62] The
variant of paragraph [61], wherein the substitution is a Ala to Cys
substitution. [63] The variant of any of paragraphs [46]-[62],
which comprises a substitution at a position corresponding to
position 265 of SEQ ID NO: 4. [64] The variant of paragraph [63],
wherein the substitution is with Gly, Pro, or Ala. [65] The variant
of paragraph [64], wherein the substitution is a Ser to Gly, Pro,
or Ala substitution. [66] The variant of any of paragraphs
[46]-[65], which comprises a substitution at a position
corresponding to position 266 of SEQ ID NO: 4. [67] The variant of
paragraph [66], wherein the substitution is with Tyr. [68] The
variant of paragraph [67], wherein the substitution is a Phe to Tyr
substitution. [69] The variant of any of paragraphs [46]-[68],
which comprises a substitution at a position corresponding to
position 391 of SEQ ID NO: 4. [70] The variant of paragraph [69],
wherein the substitution is with Asp, Trp, Val, or Asn. [71] The
variant of paragraph [70], wherein the substitution is a Thr to
Asp, Trp, Val, or Asn substitution. [72] The variant of any of
paragraphs [46]-[71], which comprises a substitution at a position
corresponding to position 393 of SEQ ID NO: 4. [73] The variant of
paragraph [72], wherein the substitution is with Asp. [74] The
variant of paragraph [73], wherein the substitution is a Ser to Asp
substitution. [75] The variant of any of paragraphs [46]-[74],
which comprises a substitution at a position corresponding to
position 394 of SEQ ID NO: 4. [76] The variant of paragraph [75],
wherein the substitution is with Pro. [77] The variant of paragraph
[76], wherein the substitution is a Ser to Pro substitution. [78]
The variant of any of paragraphs [46]-[77], wherein the catalytic
domain comprises or consists of amino acids 1 to 437 of SEQ ID NO:
26, amino acids 1 to 437 of SEQ ID NO: 28, amino acids 1 to 437 of
SEQ ID NO: 38, amino acids 1 to 437 of SEQ ID NO: 42, amino acids 1
to 437 of SEQ ID NO: 46, amino acids 1 to 437 of SEQ ID NO: 48,
amino acids 1 to 437 of SEQ ID NO: 52, amino acids 1 to 437 of SEQ
ID NO: 63, amino acids 1 to 437 of SEQ ID NO: 67, amino acids 1 to
437 of SEQ ID NO: 69, amino acids 1 to 437 of SEQ ID NO: 71, amino
acids 1 to 437 of SEQ ID NO: 73, amino acids 1 to 437 of SEQ ID NO:
75, amino acids 1 to 437 of SEQ ID NO: 77, amino acids 1 to 437 of
SEQ ID NO: 79, amino acids 1 to 437 of SEQ ID NO: 81, amino acids 1
to 437 of SEQ ID NO: 83, or amino acids 1 to 437 of SEQ ID NO: 85.
[79] The variant of any of paragraphs [46]-[78], wherein the parent
is a hybrid or chimeric polypeptide in which the carbohydrate
binding domain of the parent is replaced with a different
carbohydrate binding domain. [80] The variant of any of paragraphs
[46]-[78], which is a hybrid or chimeric polypeptide in which the
carbohydrate binding domain of the variant is replaced with a
different carbohydrate binding domain. [81] The variant of any of
paragraphs [46]-[78], wherein the parent is a fusion protein in
which a heterologous carbohydrate binding domain is fused to the
parent. [82] The variant of paragraph [81], wherein the
carbohydrate binding domain is fused to the N-terminus or the
C-terminus of the parent. [83] The variant of paragraph [81] or
[82], wherein the parent comprises or consists of SEQ ID NO: 24 or
the mature polypeptide thereof. [84] The variant of any of
paragraphs [46]-[83], which is a fusion protein in which a
heterologous carbohydrate binding domain is fused to the variant.
[85] The variant of paragraph [84], wherein the carbohydrate
binding domain is fused to the N-terminus or the C-terminus of the
variant. [86] The variant of any of paragraphs [46]-[85], which has
an increased specific performance relative to the parent. [87] An
isolated polynucleotide encoding the variant of any of
paragraphs
[1]-[86]. [88] A nucleic acid construct or expression vector
comprising the polynucleotide of paragraph [87]. [89] A host cell
comprising the polynucleotide of paragraph [87]. [90] A method of
producing a cellobiohydrolase variant, comprising: cultivating the
host cell of paragraph [89] under conditions suitable for
expression of the variant. [91] The method of paragraph [90],
further comprising recovering the variant. [92] A transgenic plant,
plant part or plant cell transformed with the polynucleotide of
paragraph [87]. [93] A method of producing the variant of any of
paragraphs [1]-[86], comprising: cultivating a transgenic plant or
a plant cell comprising a polynucleotide encoding the variant under
conditions conducive for production of the variant. [94] The method
of paragraph [93], further comprising recovering the variant [95] A
method for obtaining a cellobiohydrolase variant, comprising
introducing into a parent cellobiohydrolase an alteration at one or
more (e.g., several) positions corresponding to positions 4, 44,
45, 72, 265, 266, 391, 393 and 394 of SEQ ID NO: 4, wherein the
variant has cellobiohydrolase activity. [96] The method of
paragraph [95], further comprising recovering the variant. [97] A
composition comprising the variant of any of paragraphs [1]-[86].
[98] A whole broth formulation or cell culture composition
comprising the variant of any of paragraphs [1]-[86]. [99] A
process for degrading a cellulosic material, comprising: treating
the cellulosic material with an enzyme composition in the presence
of the variant of any of paragraphs [1]-[86]. [100] The process of
paragraph [99], wherein the cellulosic material is pretreated.
[101] The process of paragraph [99] or [100], wherein the enzyme
composition comprises one or more enzymes selected from the group
consisting of a cellulase, a GH61 polypeptide having cellulolytic
enhancing activity, a hemicellulase, a catalase, an esterase, an
expansin, a laccase, a ligninolytic enzyme, a pectinase, a
peroxidase, a protease, and a swollenin. [102] The process of
paragraph [101], wherein the cellulase is one or more enzymes
selected from the group consisting of an endoglucanase, a
cellobiohydrolase, and a beta-glucosidase. [103] The process of
paragraph [101], wherein the hemicellulase is one or more enzymes
selected from the group consisting of a xylanase, an acetylxylan
esterase, a feruloyl esterase, an arabinofuranosidase, a
xylosidase, and a glucuronidase. [104] The process of any of
paragraphs [99]-[103], further comprising recovering the degraded
cellulosic material. [105] The process of paragraph [104], wherein
the degraded cellulosic material is a sugar. [106] The process of
paragraph [105], wherein the sugar is selected from the group
consisting of glucose, xylose, mannose, galactose, and arabinose.
[107] A process for producing a fermentation product, comprising:
(a) saccharifying a cellulosic material with an enzyme composition
in the presence of the variant of any of paragraphs [1]-[86]; (b)
fermenting the saccharified cellulosic material with one or more
fermenting microorganisms to produce the fermentation product; and
(c) recovering the fermentation product from the fermentation.
[108] The process of paragraph [107], wherein the cellulosic
material is pretreated. [109] The process of paragraph [107] or
[108], wherein the enzyme composition comprises the enzyme
composition comprises one or more enzymes selected from the group
consisting of a cellulase, a GH61 polypeptide having cellulolytic
enhancing activity, a hemicellulase, a catalase, an esterase, an
expansin, a laccase, a ligninolytic enzyme, a pectinase, a
peroxidase, a protease, and a swollenin. [110] The process of
paragraph [109], wherein the cellulase is one or more enzymes
selected from the group consisting of an endoglucanase, a
cellobiohydrolase, and a beta-glucosidase. [111] The process of
paragraph [109], wherein the hemicellulase is one or more enzymes
selected from the group consisting of a xylanase, an acetylxylan
esterase, a feruloyl esterase, an arabinofuranosidase, a
xylosidase, and a glucuronidase. [112] The process of any of
paragraphs [107]-[111], wherein steps (a) and (b) are performed
simultaneously in a simultaneous saccharification and fermentation.
[113] The process of any of paragraphs [107]-[112], wherein the
fermentation product is an alcohol, an alkane, a cycloalkane, an
alkene, an amino acid, a gas, isoprene, a ketone, an organic acid,
or polyketide. [114] A process of fermenting a cellulosic material,
comprising: fermenting the cellulosic material with one or more
fermenting microorganisms, wherein the cellulosic material is
saccharified with an enzyme composition in the presence of the
variant of any of paragraphs [1]-[86]. [115] The process of
paragraph [114], wherein the fermenting of the cellulosic material
produces a fermentation product. [116] The process of paragraph
[115], further comprising recovering the fermentation product from
the fermentation. [117] The process of paragraph [115] or [116],
wherein the fermentation product is an alcohol, an alkane, a
cycloalkane, an alkene, an amino acid, a gas, isoprene, a ketone,
an organic acid, or polyketide. [118] The process of any of
paragraphs [114]-[117], wherein the cellulosic material is
pretreated before saccharification. [119] The process of any of
paragraphs [114]-[118], wherein the enzyme composition comprises
one or more enzymes selected from the group consisting of a
cellulase, a GH61 polypeptide having cellulolytic enhancing
activity, a hemicellulase, a catalase, an esterase, an expansin, a
laccase, a ligninolytic enzyme, a pectinase, a peroxidase, a
protease, and a swollenin. [120] The process of paragraph [119],
wherein the cellulase is one or more enzymes selected from the
group consisting of an endoglucanase, a cellobiohydrolase, and a
beta-glucosidase. [121] The process of paragraph [120], wherein the
hemicellulase is one or more enzymes selected from the group
consisting of a xylanase, an acetylxylan esterase, a feruloyl
esterase, an arabinofuranosidase, a xylosidase, and a
glucuronidase.
[0470] The present invention is further described by the following
examples that should not be construed as limiting the scope of the
invention.
EXAMPLES
[0471] Strains
[0472] Aspergillus oryzae strain MT3568 was used as a host for
expression of the Trichoderma reesei gene encoding
cellobiohydrolase I and a variant thereof. A. oryzae MT3568 is an
amdS (acetamidase) disrupted gene derivative of Aspergillus oryzae
JaL355 (WO 2002/40694) in which pyrG auxotrophy was restored by
disrupting the A. oryzae acetamidase (amdS) gene.
Media and Solutions
[0473] COVE sucrose plates or slants were composed of 342 g of
sucrose, 20 g of agar powder, 20 ml of COVE salt solution, and
deionized water to 1 liter. The medium was sterilized by
autoclaving at 15 psi for 15 minutes (Bacteriological Analytical
Manual, 8th Edition, Revision A, 1998). The medium was cooled to
60.degree. C. and then acetamide to 10 mM, CsCl to 15 mM, and
TRITON.RTM. X-100 (50 .mu.l/500 ml) were added.
[0474] COVE salt solution was composed of 26 g of
MgSO.sub.4.7H.sub.2O, 26 g of KCl, 26 g of KH.sub.2PO.sub.4, 50 ml
of COVE trace metals solution, and deionized water to 1 liter.
[0475] COVE trace metals solution was composed of 0.04 g of
Na.sub.2B.sub.4O.sub.7. 10H.sub.2O, 0.4 g of CuSO.sub.4.5H.sub.2O,
1.2 g of FeSO.sub.4.7H.sub.2O, 0.7 g of MnSO.sub.4.H.sub.2O, 0.8 g
of Na.sub.2MoO.sub.4.2H.sub.2O, 10 g of ZnSO.sub.4.7H.sub.2O, and
deionized water to 1 liter.
[0476] DAP-4C medium was composed of 20 g of dextrose, 10 g of
maltose, 11 g of MgSO.sub.4.7H.sub.2O, 1 g of KH.sub.2PO.sub.4, 2 g
of citric acid, 5.2 g of K.sub.3PO.sub.4.H.sub.2O, 0.5 g of yeast
extract (Difco), 1 ml of antifoam, 0.5 ml of KU6 trace metals
solution, 2.5 g of CaCO.sub.3, and deionized water to 1 liter. The
medium was sterilized by autoclaving at 15 psi for 15 minutes
(Bacteriological Analytical Manual, 8th Edition, Revision A, 1998).
Before use, 3.5 ml of sterile 50% (NH.sub.4).sub.2HPO.sub.4 and 5
ml of sterile 20% lactic acid were added per 150 ml.
[0477] G2-Gly medium was composed of 18 g of yeast extract, 24 g of
glycerol (86-88%), 1 ml of antifoam, and deionized water to 1
liter.
[0478] KU6 trace metals solution was composed of 0.13 g of
NiCl.sub.2, 2.5 g of CuSO.sub.4.5H.sub.2O, 13.9 g of
FeSO.sub.4.7H.sub.2O, 8.45 g of MnSO.sub.4.H.sub.2O, 6.8 g of
ZnCl.sub.2, 3 g of citric acid, and deionized water to 1 liter.
[0479] LB medium was composed of 10 g of Bacto-Tryptone, 5 g of
yeast extract, 10 g of sodium chloride, and deionized water to 1
liter.
[0480] LB plates were composed of 10 g of Bacto-Tryptone, 5 g of
yeast extract, 10 g of sodium chloride, 15 g of Bacto-agar, and
deionized water to 1 liter.
[0481] PDA plates were composed of potato infusion made by boiling
300 g of sliced (washed but unpeeled) potatoes in water for 30
minutes and then decanting or straining the broth through
cheesecloth. Distilled water was then added until the total volume
of the suspension was 1 liter. Then 20 g of dextrose and 20 g of
agar powder were added. The medium was sterilized by autoclaving at
15 psi for 15 minutes (Bacteriological Analytical Manual, 8th
Edition, Revision A, 1998).
[0482] TAE buffer was composed of 40 mM Tris base, 20 mM sodium
acetate, and 1 mM disodium EDTA.
[0483] YP+2% glucose medium was composed of 1% yeast extract, 2%
peptone, and 2% glucose in deionized water.
[0484] YP+2% maltose medium was composed of 10 g of yeast extract,
20 g of peptone, 20 g of maltose, and deionized water to 1
liter.
Example 1: Source of DNA Sequence Information for Aspergillus
fumigatus Cellobiohydrolase I
[0485] The genomic DNA sequence and deduced amino acid sequence of
the Aspergillus fumigatus Af293 GH7 cellobiohydrolase I gene is
shown in SEQ ID NO: 1 and SEQ ID NO: 4, respectively. Genomic
sequence information was generated by The Institute for Genomic
Research, Rockville, Md. 20850, USA and published by Nierman, W. C.
et al., 2005, Nature 438 (7071): 1151-1156. The amino acid sequence
of the full-length cellobiohydrolase I is publicly available from
the National Center for Biotechnology Information (NCBI) and
annotated as GenBank: EAL89006.1 (SEQ ID NO: 4). The cDNA sequence
and deduced amino acid sequence of the Aspergillus fumigatus
cellobiohydrolase I gene is shown in SEQ ID NO: 2 and SEQ ID NO: 4,
respectively.
[0486] Based on the publicly available amino acid sequence, a
codon-optimized synthetic gene encoding the full-length
cellobiohydrolase I was generated for Aspergillus oryzae expression
based on the algorithm developed by Gustafsson et al., 2004, Trends
in Biotechnology 22 (7): 346-353. The codon-optimized coding
sequence (SEQ ID NO: 3) was synthesized by the GENEART.RTM. Gene
Synthesis service (Life Technologies Corp., San Diego. Calif., USA)
with a 5' Bam HI restriction site, a 3' Hind III restriction site,
and a Kozac consensus sequence (CACC) situated between the start
codon and the Bam HI restriction site.
Example 2: Construction of an Aspergillus oryzae Expression Vector
Containing an Aspergillus fumigatus DNA Sequence Encoding
Cellobiohydrolase I
[0487] The ampicillin-resistant E. coli cloning vector provided by
GENEART.RTM. Gene Synthesis encoding the A. fumigatus
cellobiohydrolase I (Example 1) was digested with Barn HI and Hind
III (New England Biolabs, MA, USA) according to manufacturer's
instructions. The reaction products were isolated by 1.0% agarose
gel electrophoresis using TAE buffer where a 1606 bp product band
was excised from the gel and purified using a MinElute Gel
Extraction Kit (QIAGEN Inc., Valencia, Calif., USA).
[0488] The purified 1606 bp fragment encoding the A. fumigatus
cellobiohydrolase I was cloned into pDau109 (WO 2005/042735)
digested with Bam HI and Hind III using T4 DNA ligase (New England
Biolabs, MA, USA). The Bam HI-Hind III digested pDau109 and the Bam
HI/Hind III fragment containing the A. fumigatus cellobiohydrolase
I were mixed in a molar ratio of 1:3 (i.e., equal volumes of gel
purified products) and ligated with 50 units of T4 DNA ligase in
1.times.T4 DNA ligase buffer with 1 mM ATP and incubated at
22.degree. C. for 10 minutes.
[0489] Cloning of the A. fumigatus cellobiohydrolase I gene into
the Bam HI-Hind III digested pDau109 will result in transcription
of the A. fumigatus cellobiohydrolase I gene under the control of a
NA2-tpi double promoter. The NA2-tpi promoter is a modified
promoter from the gene encoding the Aspergillus niger neutral
alpha-amylase in which the untranslated leader has been replaced by
an untranslated leader from the gene encoding the Aspergillus
nidulans triose phosphate isomerase.
[0490] The ligation mixture was transformed into ONE SHOT.RTM.
TOP10F Chemically Competent E. coli cells according to the
manufacturer's protocol and spread onto LB plates supplemented with
0.1 mg of ampicillin per ml. After incubation at 37.degree. C.
overnight, colonies were observed growing under selection on the LB
ampicillin plates.
[0491] Insertion of the A. fumigatus cellobiohydrolase I gene into
pDau109 was verified by DNA sequencing. The isolated plasmids were
sequenced using an Applied Biosystems 3730 xl DNA Analyzer with
vector primers and A. fumigatus cellobiohydrolase I gene specific
primers, shown below, in order to determine a representative
plasmid that was free of PCR errors and contained the desired
insert.
TABLE-US-00002 Primer F-pDau109 (SEQ ID NO: 29) 5'-CCCTT GTCGA
TGCGA TGTAT C-3' Primer R-pDau109 (SEQ ID NO: 30) 5'-ATCCT CAATT
CCGTC GGTCG A-3' Primer F-pE596 (SEQ ID NO: 31) 5'-GTGAT ACACC
CGGAC AGGTG ATGTG-3' Primer R-pE596 (SEQ ID NO: 32) 5'-CCATA TCGAT
CCGAC GAGTA GGTTC-3'
[0492] An E. coli transformant containing the A. fumigatus
cellobiohydrolase I plasmid construct was cultivated in LB medium
supplemented with 0.1 mg of ampicillin per ml and plasmid DNA was
isolated using a QIAPREP.RTM. Spin Miniprep Kit. The plasmid was
designated pE596.
Example 3: Site-Directed Mutagenesis of the Aspergillus fumigatus
Cellobiohydrolase I (AC1-621)
[0493] Plasmid pE596 (Example 2) was used to generate the A.
fumigatus cellobiohydrolase I AC1-621 variant (SEQ ID NO: 25 for
the mutant DNA sequence and SEQ ID NO: 26 for the variant), wherein
a GTC codon (V44) was replaced with an ACT codon (44T).
[0494] Two synthetic primers for site-directed mutagenesis were
designed using a SOE primer design tool. Site-directed mutagenesis
of the synthetic gene encoding the wild-type A. fumigatus
cellobiohydrolase I was facilitated by PCR amplifications of pE596
using the primers and procedure described below:
TABLE-US-00003 Primer F-V44T: (SEQ ID NO: 33) 5'-AACTG GAGGT GGGTG
CACAA GACAG GCGAC TACAC CAACT GTTAC A-3' Primer R-V44T: (SEQ ID NO:
34) 5'-CTTGT GCACC CACCT CCAGT TTGCA TCGAT CAC-3'
[0495] The mutation was introduced by PCR using a PHUSION.RTM.
High-Fidelity PCR Kit (Finnzymes Oy, Espoo, Finland). The PCR
solution was composed of 10 .mu.l of 5.times.HF buffer (Finnzymes
Oy, Espoo, Finland), 4 .mu.l of dNTPs (2.5 mM), 0.5 .mu.l of
PHUSION.RTM. DNA polymerase (0.2 units/.mu.l) (Finnzymes Oy, Espoo,
Finland), 0.25 .mu.l of primer F-V44T (100 .mu.M), 0.25 .mu.l of
primer R-V44T (100 .mu.M), 5 .mu.l of template DNA (pE596, 1
ng/.mu.l), and 30 .mu.l of deionized water in a total volume of 50
.mu.l. he PCR was performed using an Applied Biosystems.RTM.
Veriti.RTM. 96 well thermal cycler programmed for 1 cycle at
98.degree. C. for 30 seconds; and 19 cycles each at 98.degree. C.
for 30 seconds, 55.degree. C. for 1 minute, and 72.degree. C. for 7
minutes. The PCR solution was then held at 8.degree. C. until
removed from the PCR machine.
[0496] Following the PCR, 10 units of Dpn I were added directly to
the PCR solution and incubated at 37.degree. C. for 1 hour. Then 1
.mu.l of the Dpn I treated PCR solution was transformed into ONE
SHOT.RTM. TOP10F' Chemically Competent E. coli cells according to
the manufacturer's protocol and spread onto LB plates supplemented
with 0.15 mg of ampicillin per ml. After incubation at 37.degree.
C. overnight, transformants were observed growing under selection
on the LB ampicillin plates. Four transformants were cultivated in
LB medium supplemented with 0.10 mg of ampicillin per ml and
plasmids were isolated using a QIAPREP.RTM. Spin Miniprep Kit
(QIAGEN Inc., Valencia, Calif., USA).
[0497] The isolated plasmids were sequenced using an Applied
Biosystems 3730xl DNA Analyzer (Applied Biosystems, Foster City,
Calif., USA) with primers F-pDau109 (SEQ ID NO: 29), R-pDau109 (SEQ
ID NO: 30), F-pE596 (SEQ ID NO: 31) and R-pE596 (SEQ ID NO: 32), in
order to determine a representative plasmid that was free of PCR
errors and contained the desired mutations.
[0498] One plasmid clone free of PCR errors and containing the GTC
(V44) to ACT (44T) mutation was chosen and designated plasmid
pE621.
Example 4: Expression of the Wild Type Aspergillus fumigatus
Cellobiohydrolase I AC1-596 and the Aspergillus fumigatus Variant
AC1-621
[0499] The expression plasmids pE596 (Example 1) and pE621 (Example
2) were transformed into Aspergillus oryzae MT3568 protoplasts
according to Christensen et al., 1988, supra and WO 2004/032648. A.
oryzae MT3568 protoplasts were prepared according to the method of
EP 0238023 B1, pages 14-15.
[0500] Transformants were purified on COVE sucrose plates without
Triton X-100 through single conidia. Spores of the transformants
were inoculated into 96 deep well plates containing 0.50 ml of
DAP-4C medium and incubated stationary at 34.degree. C. for 6
days.
[0501] Production of the wild type A. fumigatus cellobiohydrolase I
AC1-596 and the A. fumigatus cellobiohydrolase variant AC1-621 by
the transformants were analyzed from culture supernatants of the 96
deep well cultivations. Expression was verified by measuring
released reducing sugars from hydrolysis of microcrystalline
cellulose. The hydrolysis was performed in 96 well microtiter
plates (NUNC Thermo Fisher Scientific, Roskilde, Denmark) at
32.degree. C. and 1100 rpm. Each hydrolysis reaction mixture
contained 170 .mu.l of microcrystalline cellulose at 90 g/liter in
50 mM sodium acetate pH 5.0, 0.01% TRITON.RTM. X-100, 20 .mu.l of
culture supernatant, and 60 .mu.l of 50 mM sodium acetate pH 5.0,
0.01% TRITON.RTM. X-100. The plates were sealed with tape. The
hydrolysis reaction was stopped by spinning the plate at 3500 rpm
for 3 minutes. Then 12.5 .mu.l of the reaction supernatant were
added to 37.5 .mu.l MQ water in a 96 well PCR plate (Thermo Fisher
Scientific, Roskilde, Denmark). To this mixture 75 .mu.l of stop
solution was added. The stop solution was composed of 15 mg/ml
4-hydroxybenzhydrazide (Sigma Chemical Co., Inc., St. Louis, Mo.,
USA), 50 mg/ml K--Na-tartrate (Sigma Chemical Co., Inc., St. Louis,
Mo., USA) in 2% (w/v) NaOH. The plate was sealed with a lid and the
mixture was incubated at 95.degree. C. for 10 minutes and 5 minutes
at 20.degree. C. Then 100 .mu.l was transferred to a microtiter
plate and absorbance at 410 nm was measured using a SPECTRAMAX.RTM.
Plus 384 (Molecular Devices, Sunnyvale, Calif., USA). The
concentration of reducing sugar was proportional to the absorbance
at 410 nm of the oxidized 4-hydroxybenzhydrazide. The reducing
sugar content in the culture supernatants was measured by adding 1
.mu.l of culture supernatant to a mixture of 75 .mu.l of stop
solution and 49 .mu.l of milliQ water in a 96 well PCR plate. The
plate was sealed with a lid and the mixture was incubated at
95.degree. C. for 10 minutes and 5 minutes at 20.degree. C. Then
100 .mu.l was transferred to a microtiter plate and the absorbance
at 410 nm was measured. The absorbance at 410 nm from the cell
culture supernatant was subtracted from the absorbance at 410 nm of
the hydrolysis reaction, to measure the amount of reducing sugar
released by the enzymes.
[0502] Based on the level of hydrolysis of the microcrystalline
cellulose one transformant expressing the wild type A. fumigatus
cellobiohydrolase I was selected and designated A. oryzae AC1-596.
Based on the level of hydrolysis of the microcrystalline cellulose
one transformant expressing the A. fumigatus cellobiohydrolase I
variant AC1-621 was selected and designated A. oryzae AC1-621.
[0503] For larger scale production, A. oryzae AC1-596 or A. oryzae
AC1-621 spores were spread onto COVE sucrose slants and incubated
for five days at 37.degree. C. The confluent spore slants were
washed twice with 5 ml MQ water with 0.01% TWEEN.RTM. 20. The spore
suspensions were then used to inoculate a 500 ml flask containing
150 ml of G2-Gly medium. The pre-culture was incubated at
30.degree. C. with constant shaking at 200 rpm. After one day, the
pre-culture was used to inoculate four 500 ml flasks containing 200
ml of DAP-4C medium. At day four post-inoculation, the culture
broths were collected by filtration through a bottle top MF75 Supor
MachV 0.2 .mu.m PES filter.
Example 5: Site-Directed Mutagenesis of the Aspergillus fumigatus
Cellobiohydrolase I (AC1-625)
[0504] Plasmid pE596 (Example 2) was used to generate the A.
fumigatus cellobiohydrolase I AC1-625 variant (SEQ ID NO: 27 for
the mutant DNA sequence and SEQ ID NO: 28 for the variant), a TCC
codon (S265) was replaced with a TCC codon (265P).
[0505] Two synthetic primers for site-directed mutagenesis were
designed using a SOE primer design tool. Site-directed mutagenesis
of the synthetic gene encoding the wild-type A. fumigatus
cellobiohydrolase I was facilitated by PCR amplifications of pE596
using the primers and procedure described below:
TABLE-US-00004 Primer F-S265P: (SEQ ID NO: 35) 5'-CCCGA CGGCT GTGAC
TTCAA CCCTT TCAGG CAGGG CAACA AAACA TT-3' Primer R-S265P: (SEQ ID
NO: 36) 5'-GTTGA AGTCA CAGCC GTCGG GGTCA CACGT AC-3'
[0506] The mutation was introduced by PCR using a PHUSION.RTM.
High-Fidelity PCR Kit (New England Biolabs, MA, USA). The PCR
solution was composed of 10 .mu.l of 5.times.HF buffer (New England
Biolabs, MA, USA), 4 .mu.l of dNTPs (2.5 mM), 0.5 .mu.l of
PHUSION.RTM. High-Fidelity DNA polymerase (0.2 units/.mu.l) (New
England Biolabs, MA, USA), 0.25 .mu.l of primer F-S265P (100
.mu.M), 0.25 .mu.l of primer R-S265P (100 .mu.M), 5 .mu.l of
template DNA (pE596, 1 ng/.mu.l), and 30 .mu.l of deionized water
in a total volume of 50 .mu.l. he PCR was performed using an
Applied Biosystems.RTM. Veriti.RTM. 96 well thermal cycler
programmed for 1 cycle at 98.degree. C. for 30 seconds; and 19
cycles each at 98.degree. C. for 30 seconds, 55.degree. C. for 1
minute, and 72.degree. C. for 7 minutes. The PCR solution was then
held at 8.degree. C. until removed from the PCR machine.
[0507] Following the PCR, 10 units of Dpn I were added directly to
the PCR solution and incubated at 37.degree. C. for 1 hour. Then 1
.mu.l of the Dpn I treated PCR solution was transformed into ONE
SHOT.RTM. TOP10F' Chemically Competent E. coli cells according to
the manufacturer's protocol and spread onto LB plates supplemented
with 0.15 mg of ampicillin per ml. After incubation at 37.degree.
C. overnight, transformants were observed growing under selection
on the LB ampicillin plates. Four transformants were cultivated in
LB medium supplemented with 0.10 mg of ampicillin per ml and
plasmids were isolated using a QIAPREP.RTM. Spin Miniprep Kit.
[0508] The isolated plasmids were sequenced using an Applied
Biosystems 3730xl DNA Analyzer with primers F-pDau109 (SEQ ID NO:
29), R-pDau109 (SEQ ID NO: 30), F-pE596 (SEQ ID NO: 31) and R-pE596
(SEQ ID NO: 32), in order to determine a representative plasmid
that was free of PCR errors and contained the desired
mutations.
[0509] One plasmid clone free of PCR errors and containing the TCC
(S265) to CCT (265P) mutation was chosen and designated plasmid
pE625.
Example 6: Expression of the Wild Type Aspergillus fumigatus
Cellobiohydrolase I AC1-596 and the Aspergillus fumigatus Variant
AC1-625
[0510] The expression plasmids pE596 (Example 1) and pE625 (Example
5) were transformed into Aspergillus oryzae MT3568 protoplasts
according to Christensen et al., 1988, supra and WO 2004/032648. A.
oryzae MT3568 protoplasts were prepared according to the method of
EP 0238023 B1, pages 14-15.
[0511] Transformants were purified on COVE sucrose plates without
Triton X-100 through single conidia. Spores of the transformants
were inoculated into 96 deep well plates containing 0.50 ml of
DAP-4C medium and incubated stationary at 34.degree. C. for 6
days.
[0512] Production of the wild type A. fumigatus cellobiohydrolase I
AC1-596 and the A. fumigatus cellobiohydrolase variant AC1-625 by
the transformants were analyzed from culture supernatants of the 96
deep well cultivations. Expression was verified by measuring
released reducing sugars from hydrolysis of microcrystalline
cellulose. The hydrolysis was performed in 96 well microtiter
plates (NUNC Thermo Fisher Scientific, Roskilde, Denmark) at
32.degree. C. and 1100 rpm. Each hydrolysis reaction mixture
contained 170 .mu.l of microcrystalline cellulose at 90 g/liter in
50 mM sodium acetate pH 5.0, 0.01% TRITON.RTM. X-100, 20 .mu.l of
culture supernatant, and 60 .mu.l of 50 mM sodium acetate pH 5.0,
0.01% TRITON.RTM. X-100. The plates were sealed with tape. The
hydrolysis reaction was stopped by spinning the plate at 3500 rpm
for 3 minutes. Then 12.5 .mu.l of the reaction supernatant were
added to 37.5 .mu.l MQ water in a 96 well PCR plate (Thermo Fisher
Scientific, Roskilde, Denmark). To this mixture 75 .mu.l of stop
solution was added. The stop solution was composed of 15 mg/ml
4-hydroxybenzhydrazide (Sigma Chemical Co., Inc., St. Louis, Mo.,
USA), 50 mg/ml K--Na-tartrate (Sigma Chemical Co., Inc., St. Louis,
Mo., USA) in 2% (w/v) NaOH. The plate was sealed with a lid and the
mixture was incubated at 95.degree. C. for 10 minutes and 5 minutes
at 20.degree. C. Then 100 .mu.l was transferred to a microtiter
plate and absorbance at 410 nm was measured using a SPECTRAMAX.RTM.
Plus 384 (Molecular Devices, Sunnyvale, Calif., USA). The
concentration of reducing sugar was proportional to the absorbance
at 410 nm of the oxidized 4-hydroxybenzhydrazide. The reducing
sugar content in the culture supernatants was measured by adding 1
.mu.l of culture supernatant to a mixture of 75 .mu.l of stop
solution and 49 .mu.l of milliQ water in a 96 well PCR plate. The
plate was sealed with a lid and the mixture was incubated at
95.degree. C. for 10 minutes and 5 minutes at 20.degree. C. Then
100 .mu.l was transferred to a microtiter plate and the absorbance
at 410 nm was measured. The absorbance at 410 nm from the cell
culture supernatant was subtracted from the absorbance at 410 nm of
the hydrolysis reaction, to measure the amount of reducing sugar
released by the enzymes.
[0513] Based on the level of hydrolysis of the microcrystalline
cellulose one transformant expressing the wild type A. fumigatus
cellobiohydrolase I was selected and designated A. oryzae AC1-596.
Based on the level of hydrolysis of the microcrystalline cellulose
one transformant expressing the A. fumigatus cellobiohydrolase I
variant AC1-625 was selected and designated A. oryzae AC1-625.
[0514] For larger scale production, A. oryzae AC1-596 or A. oryzae
AC1-625 spores were spread onto COVE sucrose slants and incubated
for five days at 37.degree. C. The confluent spore slants were
washed twice with 5 ml MQ water with 0.01% TWEEN.RTM. 20. The spore
suspensions were then used to inoculate a 500 ml flask containing
150 ml of G2-Gly medium. The pre-culture was incubated at
30.degree. C. with constant shaking at 200 rpm. After one day, the
pre-culture was used to inoculate four 500 ml flasks containing 200
ml of DAP-4C medium. At day four post-inoculation, the culture
broths were collected by filtration through a bottle top MF75 Supor
MachV 0.2 .mu.m PES filter.
Example 7: Purification of the Aspergillus fumigatus Wild-Type
Cellobiohydrolase I and Aspergillus fumigatus Cellobiohydrolase I
Variants AC1-621 and AC1-625
[0515] The broths of Examples 4 & 6 were filtered using a 0.22
.mu.m PES filter (Nalge Nunc International Corp., Rochester, N.Y.,
USA), followed by addition of ammonium sulphate to a concentration
of 1.8 M and another filtration.
[0516] Each filtrate was purified according to the following
procedure. The filtrate was loaded onto a Phenyl SEPHAROSE.RTM. 6
Fast Flow column (high sub) (GE Healthcare, Piscataway, N.J., USA)
equilibrated with 1.8 M ammonium sulphate, 25 mM HEPES pH 7.0.
After a wash with 2 CV 1.8 M ammonium sulphate followed by 1 CV
0.54 M ammonium sulphate, the bound proteins were batch eluted with
25 mM HEPES pH 7.0.
[0517] The elution of the protein was monitored at 280 nm and
fractions were collected and analyzed by SDS-PAGE using 12-well
NUPAGE.RTM. 4-12% Bis-Tris gel (GE Healthcare, Piscataway, N.J.,
USA). The fractions were pooled based on SDS-PAGE as above and
applied to a SEPHADEX.TM. G-25 (medium) column (GE Healthcare,
Piscataway, N.J., USA) equilibrated with 25 mM MES pH 6.0.
Fractions were collected, analyzed by SDS-PAGE as above, and
pooled.
[0518] The pooled fractions were applied to a 53 ml RESOURCE.TM.
15Q column (GE Healthcare, Piscataway, N.J., USA) equilibrated with
25 mM MES pH 6.0 and bound proteins were eluted with a linear
100-200 mM sodium chloride gradient for 2 CV followed by 1 CV 1 M
sodium chloride. The elution of the protein was monitored at 280 nm
and fractions with high absorbance at 280 nm were analyzed on
SDS-PAGE. Fractions with high content of cellobiohydrolase I were
pooled.
Example 8: Activity Measurement on Microcrystalline Cellulose of
the Aspergillus fumigatus Cellobiohydrolase I Variant AC1-621
[0519] The activity of the purified cellobiohydrolase I AC1-621
variant (Example 7) was compared to the purified A. fumigatus
wild-type cellobiohydrolase I (Example 7) using microcrystalline
cellulose (AVICEL.RTM. PH101; Sigma-Aldrich, St. Louis, Mo., USA)
as a substrate.
[0520] The purified cellobiohydrolase I variant was diluted in 50
mM sodium acetate, 2 mM CaCl.sub.2 pH 5. The diluted
cellobiohydrolase I variant was added to each well (microwell plate
96F 26960 Thermo scientific). Washed AVICEL was added to each well
and the microtiter plate was quickly transferred to a thermomixer
(eppendorf) and incubated for 24 hours at 1100 rpm and 50.degree.
C. or 60.degree. C. The final concentration of cellobiohydrolase I
variant in the reaction was 3 .mu.M, the concentration of
.beta.-glucosidase was 0.15 .mu.M and the concentration of AVICEL
was 80 g/L. The reaction was stopped by centrifugation at 3500 rpm
for 3 min at 5.degree. C. (Hereaus multifuge 3 S-R). The
supernatants diluted and transferred to PCR sample tubes
(Thermoscientific 0.2 ml non-skirtet 96-well PCR plate AB0600).
PAHBAH (4-hydroxy-benzhydrazid) (Sigma, H 9882) was dissolved in
buffer (0.18 M K--Na-tartrate (Merck, 1.08087) and 0.5 M NaOH) to
make a 15 mg/ml solution. 75 .mu.L of the PAHBAH solution was added
to 50 .mu.L of the supernatants in the PCR samples tubes.
[0521] The PCR sample tubes were placed in a Peltier Thermal Cycler
and incubated at 95.degree. C. for 10 min and 20.degree. C. for 5
min. Following incubation, 100 .mu.L were transferred to a 96 well
microtiter plate (microwell plate 96F 26960 Thermo scientific) and
the absorbance was measured at 410 nm. For each run a standard was
included. The standard used was glucose diluted in 50 mM sodium
acetate, 2 mM CaCl.sub.2 pH 5 to a concentration of 0.008, 0.016,
0.0312, 0.0625, 0.125, 0.25, 0.5 mM. In addition to the standard, a
blank with .beta.-glucosidase (without cellobiohydrolase) for each
run was included. For all the measurement, the blank measurement
was subtracted. The absorbance data were normalized to glucose
concentration using the standards.
[0522] The results demonstrated that the A. fumigatus variant
AC1-621 (V44T) had an approximately 15% increase activity toward
microcrystalline cellulose compared to the wild-type parent
cellobiohydrolase at 50.degree. C. (FIG. 1) and an increase of 13%
compared to the wild-type parent cellobiohydrolase at 60.degree. C.
(FIG. 2).
Example 9: Pretreated Corn Stover Hydrolysis Assay
[0523] Corn stover was pretreated at the U.S. Department of Energy
National Renewable Energy Laboratory (NREL) using 1.4 wt % sulfuric
acid at 165.degree. C. and 107 psi for 8 minutes. The
water-insoluble solids in the pretreated corn stover (PCS)
contained 56.5% cellulose, 4.6% hemicellulose, and 28.4% lignin.
Cellulose and hemicellulose were determined by a two-stage sulfuric
acid hydrolysis with subsequent analysis of sugars by high
performance liquid chromatography using NREL Standard Analytical
Procedure #002. Lignin was determined gravimetrically after
hydrolyzing the cellulose and hemicellulose fractions with sulfuric
acid using NREL Standard Analytical Procedure #003.
[0524] Unmilled, unwashed PCS (whole slurry PCS) was prepared by
adjusting the pH of the PCS to 5.0 by addition of 10 M NaOH with
extensive mixing, and then autoclaving for 20 minutes at
120.degree. C. The dry weight of the whole slurry PCS was 29%.
[0525] A 96-well plate was generated by machining a teflon plate of
depth 1/4 inch with 96, cone-shaped wells, diameter 1/4 inch at the
upper surface and diameter 1/8 inch at the lower surface. The
center of each well was at an equivalent position to the center of
a corresponding well in a standard 96-well microtiter plate,
approximately 23/64 inch on center. The resulting volume of each
well was approximately 135 .mu.l. This 96-well aluminum plate is
hereinafter referred to as the "fill plate". The pH-adjusted corn
stover was used to fill the holes in the fill plate by applying a
suitable volume of the corn stover to the upper surface of the
plate, then using a spatula to spread the material over the surface
and into the holes. Holes were deemed sufficiently full when corn
stover was extruded through the hole in the bottom surface, forming
noodle-like tubes. A MULTISCREEN.RTM. Column Loader Scraper
(Millipore) held perpendicular to the fill plate surface was used
to scrape excess corn stover from the top and bottom surfaces of
the fill plate, leaving the surfaces of the corn stover in each
well flush with the surfaces of the fill plate. The fill plate was
then placed on the top of a 2.2 ml deep well plate (Axygen, Union
City, Calif., USA) with the top surface adjacent to the open end of
the well plate (e.g. the top of the well plate), and the wells
aligned with the corn stover-filled holes in the fill plate. The
fill plate was secured in this position, and the assembly
centrifuged at 2500 rpm (1350.times.g) for 5 minutes in a Sorvall
Legend RT+(Thermo Scientific, Waltham, Mass., USA). Following
centrifugation, the corn stover had been transferred to the deep
well plate. A 3 mm glass bead (Fisher Scientific, Waltham, Mass.,
USA) was placed in each well for mixing.
[0526] The hydrolysis of PCS was conducted in a total reaction
volume of 0.2 ml. The hydrolysis was performed with 50 mg of
insoluble PCS solids containing 50 mM sodium acetate pH 5.0 buffer
containing 1 mM manganese sulfate and various protein loadings of
various enzyme compositions (expressed as mg protein per gram of
cellulose). Enzyme compositions were prepared and then added
simultaneously to all wells in a volume ranging from 20 .mu.l to 50
.mu.l, for a final volume of 0.2-0.50 ml in each reaction. The
plate was then sealed using an ALPS-300.TM. plate heat sealer
(Abgene, Epsom, United Kingdom), mixed thoroughly, and incubated at
a specific temperature for 72 hours.
[0527] Following hydrolysis, samples were filtered using a 0.45
.mu.m MULTISCREEN.RTM. 96-well filter plate (Millipore, Bedford,
Mass., USA) and filtrates analyzed for sugar content as described
below. When not used immediately, filtered aliquots were frozen at
-20.degree. C. The sugar concentrations of samples diluted in 0.005
M H.sub.2SO.sub.4 were measured using a 4.6.times.250 mm
AMINEX.RTM. HPX-87H column (Bio-Rad Laboratories, Inc., Hercules,
Calif., USA) by elution with 0.05% w/w benzoic acid-0.005 M
H.sub.2SO.sub.4 at 65.degree. C. at a flow rate of 0.6 ml per
minute, and quantitation by integration of the glucose, cellobiose,
and xylose signals from refractive index detection
(CHEMSTATION.RTM., AGILENT.RTM. 1100 HPLC, Agilent Technologies,
Santa Clara, Calif., USA) calibrated by pure sugar samples. The
resultant glucose and cellobiose equivalents were used to calculate
the percentage of cellulose conversion for each reaction.
[0528] Glucose and cellobiose were measured individually. Measured
sugar concentrations were adjusted for the appropriate dilution
factor. The net concentrations of enzymatically-produced sugars
from unwashed PCS were determined by adjusting the measured sugar
concentrations for corresponding background sugar concentrations in
unwashed PCS at zero time point. All HPLC data processing was
performed using MICROSOFT EXCEL.TM. software (Microsoft, Richland,
Wash., USA).
[0529] The degree of glucose conversion to glucose was calculated
using the following equation: % cellulose conversion=(glucose
concentration)/(glucose concentration in a limit digest).times.100.
In order to calculate % glucose conversion, a 100% conversion point
was set based on a cellulase control (100 mg of T. reesei cellulase
supplemented with Thermoascus aurantiacus GH61A polypeptide,
Aspergillus fumigatus GH10 xylanase (xyn3), and Talaromyces
emersonii beta-xylosidase per gram cellulose). Quadruplicate data
points were averaged and standard deviation was calculated.
Example 10: Preparation of an Enzyme Composition without
Cellobiohydrolase I
[0530] The Talaromyces leycettanus GH6 cellobiohydrolase II
(GENESEQP:AZY49446) was prepared recombinantly in Aspergillus
oryzae as described in WO 2012/103288. The filtered broth of the
Talaromyces leycettanus GH6 cellobiohydrolase II was concentrated
and buffer exchanged into 20 mM Tris pH 8.0 using a 400 ml
SEPHADEX.TM. G-25 column (GE Healthcare, United Kingdom). The
fractions were pooled, and 3 M ammonia sulfate, 20 mM Tris pH 8.0
was added to the desalted protein to a final concentration of 1.2 M
ammonia sulfate, 20 mM Tris pH 8.0. The protein was loaded onto a
Phenyl Sepharose.TM. 6 Fast Flow column (high sub) (GE Healthcare,
Piscataway, N.J., USA) equilibrated in 20 mM Tris pH 8.0 with 1.2 M
ammonium sulfate, and bound proteins were eluted with 20 mM Tris pH
8.0 with no ammonium sulfate. Fractions were analyzed by 8-16%
Tris-HCl SDS-PAGE gels (Bio-Rad, Hercules, Calif., USA), and
pooled. The pooled protein was buffer exchanged into 20 mM MES pH
6.0 using a Vivaflow 200 with 10 kDa molecular weight cut-off
tangential flow membrane (Sartorius, Bohemia, N.Y., USA).
[0531] The Trichoderma reesei GH5 endoglucanase II
(GENESEQP:AZ104858) was prepared recombinantly according to WO
2011/057140 using Aspergillus oryzae as a host. The filtered broth
of the T. reesei endoglucanase II was desalted and buffer-exchanged
into 20 mM Tris pH 8.0 using using a tangential flow concentrator
(Pall Filtron, Northborough, Mass., USA) equipped with a 10 kDa
polyethersulfone membrane (Pall Filtron, Northborough, Mass.,
USA).
[0532] Thermoascus aurantiacus CGMCC 0583 GH61A polypeptide having
cellulolytic enhancing activity (GENESEQP:AEC05922) was
recombinantly prepared according to WO 2005/074656 using
Aspergillus oryzae JaL250 as a host. The broth was filtered using a
0.22 .mu.m EXPRESS.TM. Plus Membrane (Millipore, Bedford, Mass.,
USA).
[0533] The Aspergillus fumigatus GH10 xylanase (xyn3)
(GENESEQP:AZ104884) was prepared recombinantly according to WO
2006/078256 using Aspergillus oryzae BECh2 (WO 2000/39322) as a
host. The filtered broth of the A. fumigatus xylanase was desalted
and buffer-exchanged into 50 mM sodium acetate pH 5.0 using a
HIPREP.RTM. 26/10 Desalting Column (GE Healthcare, Piscataway,
N.J., USA).
[0534] The Aspergillus fumigatus Cel3A beta-glucosidase 4M variant
(GENESEQP:AZU67153) was recombinantly prepared according to WO
2012/044915. The filtered broth of Aspergillus fumigatus Cel3A
beta-glucosidase 4M was concentrated and buffer exchanged using a
tangential flow concentrator (Pall Filtron, Northborough, Mass.,
USA) equipped with a 10 kDa polyethersulfone membrane (Pall
Filtron, Northborough, Mass., USA) with 50 mM sodium acetate pH 5.0
containing 100 mM sodium chloride. Protein concentration was using
4-nitrophenyl-beta-d-glucopyranoside (Sigma Chemical Co., Inc., St.
Louis, Mo., USA) as a substrate and Aspergillus fumigatus Cel3A
beta-glucosidase 4M 280 as a protein standard purified according to
WO 2012/044915 with the protein concentration determined using the
theoretic extinction coefficient and the absorbance of the protein
at 280 nm. The 4-nitrophenyl-beta-d-glucopyranoside (pNPG) was
performed as follows: pNPG was dissolved in DMSO to make 100 mM
stock solution. The 100 mM pNPG stock solution was diluted
100.times. in 50 mM sodium acetate buffer pH 5 with 0.01% Tween 20
to 1 mM pNPG containing 50 mM sodium acetate buffer pH 5 with 0.01%
Tween 20. The protein was diluted at several concentrations in 50
mM sodium acetate buffer pH 5 with 0.01% Tween 20. Then, 20 ul of
diluted protein was added to 100 ul of 1 mM pNPG containing 50 mM
sodium acetate buffer pH 5 with 0.01% Tween 20. The reactions were
incubated at 40.degree. C. for 20 minutes, and reactions were
stopped with 50 ul 1M sodium carbonate buffer pH 10. The absorbance
was measured for pNP production at 405 nm.
[0535] The Talaromyces emersonii CBS 393.64 beta-xylosidase
(GENESEQP:AZ104896) was prepared recombinantly according to
Rasmussen et al., 2006, Biotechnology and Bioengineering 94:
869-876 using Aspergillus oryzae JaL355 as a host (WO 2003/070956).
The filtered broth was concentrated and desalted with 50 mM sodium
acetate pH 5.0 using a tangential flow concentrator equipped with a
10 kDa polyethersulfone membrane (Pall Filtron, Northborough,
Mass., USA).
[0536] The protein concentration for each of the monocomponents
described above except the Aspergillus fumigatus Cel3A
beta-glucosidase 4M variant was determined using a Microplate
BOA.TM. Protein Assay Kit (Thermo Fischer Scientific, Waltham,
Mass., USA) in which bovine serum albumin was used as a protein
standard. An enzyme composition was prepared composed of each
monocomponent as follows: 39.7% Talaromyces leycettanus GH6
cellobiohydrolase II, 15.9% Trichoderma reesei GH5 endoglucanase
II, 23.8% Thermoascus aurantiacus GH61A polypeptide, 7.9%
Aspergillus fumigatus GH10 xylanase, 7.9% Aspergillus fumigatus
beta-glucosidase, and 4.8% Talaromyces emersonii beta-xylosidase.
The enzyme composition is designated herein as "cellulolytic enzyme
composition without cellobiohydrolase I".
Example 11: Comparison of the Effect of Aspergillus fumigatus
Cellobiohydrolase I AC1-621 Variant Polypeptide Against Aspergillus
fumigatus Cellobiohydrolase I AC1-596 Wild-Type Polypeptide on the
Hydrolysis of Unwashed PCS by a Cellulase Enzyme Composition
[0537] The Aspergillus fumigatus cellobiohydrolase I AC1-621
variant polypeptide (Example 7) was added to the cellulolytic
enzyme composition without cellobiohydrolase I (Example 10) at
35.degree. C., 50.degree. C., 55.degree. C., 60.degree. C., and
65.degree. C. using unwashed PCS as a substrate, and compared
against the Aspergillus fumigatus cellobiohydrolase I AC1-596
wild-type polypeptide added to the cellulolytic enzyme composition
without cellobiohydrolase I. Each cellobiohydrolase I was added
individually at 3.33 mg enzyme protein per g cellulose to 5.67 mg
enzyme protein of the cellulase enzyme composition without
cellobiohydrolase I per g cellulose.
[0538] The assay was performed as described in Example 9. The
reactions with unwashed PCS (20% total solids) were conducted for
72 hours at 35.degree. C., 50.degree. C., 55.degree. C., 60.degree.
C., and 65.degree. C. in 71 mM sodium acetate pH 5.0 buffer
containing 1 mM manganese sulfate. All reactions were performed in
quadruplicate and shaking at 200 rpm throughout the hydrolysis.
[0539] The results shown in FIG. 3 demonstrated that the cellulase
enzyme composition containing the Aspergillus fumigatus
cellobiohydrolase I AC1-621 variant polypeptide had significantly
higher cellulose conversion than the cellulase enzyme composition
that included Aspergillus fumigatus cellobiohydrolase I AC1-596
wild-type polypeptide at 35.degree. C., 50.degree. C., 55.degree.
C., and 60.degree. C.
Example 12: Comparison of the Effect of Aspergillus fumigatus
Cellobiohydrolase I AC1-621 Variant Polypeptide and Aspergillus
fumigatus Cellobiohydrolase I AC1-625 Variant Polypeptide Against
Aspergillus fumigatus Cellobiohydrolase I AC1-596 Wild-Type
Polypeptide on the Hydrolysis of Unwashed PCS by a Cellulase Enzyme
Composition
[0540] The Aspergillus fumigatus cellobiohydrolase I AC1-621
variant polypeptide and cellobiohydrolase I AC1-625 (Example 7)
were added to the cellulolytic enzyme composition without
cellobiohydrolase I (Example 10) at 35.degree. C., 50.degree. C.
and 60.degree. C. using unwashed PCS as a substrate, and compared
against the Aspergillus fumigatus cellobiohydrolase I AC1-596
wild-type polypeptide added to the cellulolytic enzyme composition
without cellobiohydrolase I. Each cellobiohydrolase I was added
individually at 3.33 mg enzyme protein per g cellulose to 5.67 mg
enzyme protein of the cellulase enzyme composition without
cellobiohydrolase I per g cellulose.
[0541] The assay was performed as described in Example 9. The
reactions with unwashed PCS (20% total solids) were conducted for
24, 48, and 72 hours at 35.degree. C., 50.degree. C. and 60.degree.
C. in 71 mM sodium acetate pH 5.0 buffer containing 1 mM manganese
sulfate. All reactions were performed in quadruplicate and shaking
at 200 rpm throughout the hydrolysis.
[0542] The results for 35.degree. C., 50.degree. C., and 60.degree.
C. are shown in FIGS. 4, 5, and 6, respectively. The results
demonstrated that the cellulase enzyme composition containing the
Aspergillus fumigatus cellobiohydrolase I AC1-621 variant
polypeptide had significantly higher cellulose conversion than the
cellulase enzyme composition that included Aspergillus fumigatus
cellobiohydrolase I AC1-596 wild-type polypeptide at 35.degree. C.
for 24, 48, and 72 hours, 50.degree. C. for 24, 48, and 72 hours,
and 60.degree. C. for 72 hours. In addition, the results
demonstrated that the cellulase enzyme composition containing the
Aspergillus fumigatus cellobiohydrolase I AC1-625 variant
polypeptide had significantly higher cellulose conversion than the
cellulase enzyme composition that included Aspergillus fumigatus
cellobiohydrolase I AC1-596 wild-type polypeptide at 35.degree. C.
for 48 and 72 hours, 50.degree. C. for 48 and 72 hours, and
60.degree. C. for 24 and 72 hours.
Example 13: Determination of Td by Differential Scanning
Calorimetry of the Aspergillus fumigatus Cellobiohydrolase I
AC1-625 Variant and Aspergillus fumigatus Cellobiohydrolase I
AC1-596 Wild-Type Polypeptide
[0543] The thermostability of the Aspergillus fumigatus wild-type
cellobiohydrolase I AC1-596 and cellobiohydrolase I AC1-625 variant
was determined by Differential Scanning calorimetry (DSC) using a
VP-Capillary Differential Scanning calorimeter (MicroCal Inc.,
Piscataway, N.J., USA). The thermal denaturation temperature, Td
(.degree. C.), was taken as the top of denaturation peak (major
endothermic peak) in thermograms (Cp vs. T) obtained after heating
enzyme solutions (approx. 1 mg/ml) in 50 mM sodium acetate pH 5.0
at a constant programmed heating rate of 200 K/hour.
[0544] Sample- and reference-solutions (approx. 0.2 ml) were loaded
into the calorimeter (reference: buffer without enzyme) from
storage conditions at 10.degree. C. and thermally pre-equilibrated
for 20 minutes at 20.degree. C. prior to DSC scan from 20.degree.
C. to 100.degree. C. Denaturation temperatures were determined at
an accuracy of approximately +/-1.degree. C.
[0545] The results demonstrated that the Aspergillus fumigatus
wild-type cellobiohydrolase I has a Td of 71.degree. C. compared to
74.degree. C. for the cellobiohydrolase I AC1-625 variant.
Example 14: Site-Directed Mutagenesis of the Aspergillus fumigatus
Cellobiohydrolase I (AC1-870)
[0546] Plasmid pE596 (Example 2) was used to generate the A.
fumigatus cellobiohydrolase I AC1-870 variant (SEQ ID NO: 37 for
the mutant DNA sequence and SEQ ID NO: 38 for the variant), an ACT
codon (T391) was replaced with a GAT codon (391D), a TCG codon
(S393) was replaced with a GAT codon (393D) and a TCC codon (S394)
was replaced with a CCT codon (394P).
[0547] Two synthetic primers for site-directed mutagenesis were
designed using a SOE primer design tool. Site-directed mutagenesis
of the synthetic gene encoding the wild-type A. fumigatus
cellobiohydrolase I was facilitated by PCR amplifications of pE596
using the primers and procedure described below:
TABLE-US-00005 Primer F-T391D-S393D-S394P: (SEQ ID NO: 39) 5'-GTTGG
ATTCC AACTA CCCCA CCGAT GCCGA TCCTA CGACA CCGGG TGTCG CACG-3'
Primer R-T391D-S393D-S394P: (SEQ ID NO: 40) 5'-GGTGG GGTAG TTGGA
ATCCA ACCAG AGCAT GTTG-3'
[0548] The mutation was introduced by PCR using a PHUSION.RTM.
High-Fidelity PCR Kit (New England Biolabs, MA, USA). The PCR
solution was composed of 10 .mu.l of 5.times.HF buffer (New England
Biolabs, MA, USA), 4 .mu.l of dNTPs (2.5 mM), 0.5 .mu.l of
PHUSION.RTM. High-Fidelity DNA polymerase (0.2 units/.mu.l) (New
England Biolabs, MA, USA), 0.25 .mu.l of primer F-T391D-S393D-S394P
(100 .mu.M), 0.25 .mu.l of primer R-T391D-S393D-S394P (100 .mu.M),
5 .mu.l of template DNA (pE596, 1 ng/.mu.l), and 30 .mu.l of
deionized water in a total volume of 50 .mu.l. he PCR was performed
using an Applied Biosystems.RTM. Veriti.RTM. 96 well thermal cycler
programmed for 1 cycle at 98.degree. C. for 30 seconds; and 19
cycles each at 98.degree. C. for 30 seconds, 55.degree. C. for 1
minute, and 72.degree. C. for 7 minutes. The PCR solution was then
held at 8.degree. C. until removed from the PCR machine.
[0549] Following the PCR, 10 units of Dpn I were added directly to
the PCR solution and incubated at 37.degree. C. for 1 hour. Then 1
.mu.l of the Dpn I treated PCR solution was transformed into ONE
SHOT.RTM. TOP10F' Chemically Competent E. coli cells according to
the manufacturer's protocol and spread onto LB plates supplemented
with 0.15 mg of ampicillin per ml. After incubation at 37.degree.
C. overnight, transformants were observed growing under selection
on the LB ampicillin plates. Four transformants were cultivated in
LB medium supplemented with 0.10 mg of ampicillin per ml and
plasmids were isolated using a QIAPREP.RTM. Spin Miniprep Kit.
[0550] The isolated plasmids were sequenced using an Applied
Biosystems 3730xl DNA Analyzer with primers F-pDau109 (SEQ ID NO:
28), R-pDau109 (SEQ ID NO: 29), F-pE596 (SEQ ID NO: 30) and R-pE596
(SEQ ID NO: 31), in order to determine a representative plasmid
that was free of PCR errors and contained the desired
mutations.
[0551] One plasmid clone free of PCR errors and containing the ACT
(T391) to GAT (391D), the TCG (S393) to GAT (393D) and the TCC
(S394) to CCT (394P) mutations was chosen and designated plasmid
pE870.
Example 15: Expression of the Aspergillus fumigatus Variant
AC1-870
[0552] The expression plasmid pE870 (Example 14) was transformed
into Aspergillus oryzae MT3568 protoplasts according to Christensen
et al., 1988, supra and WO 2004/032648. A. oryzae MT3568
protoplasts were prepared according to the method of EP 0238023 B1,
pages 14-15.
[0553] Transformants were purified on COVE sucrose plates without
Triton X-100 through single conidia. Spores of the transformants
were inoculated into 96 deep well plates containing 0.50 ml of
DAP-4C medium and incubated stationary at 34.degree. C. for 6
days.
[0554] Production of the A. fumigatus cellobiohydrolase variant
AC1-870 by the transformants were analyzed from culture
supernatants of the 96 deep well cultivations. Expression was
verified by measuring released reducing sugars from hydrolysis of
microcrystalline cellulose. The hydrolysis was performed in 96 well
microtiter plates (NUNC Thermo Fisher Scientific, Roskilde,
Denmark) at 32.degree. C. and 1100 rpm. Each hydrolysis reaction
mixture contained 170 .mu.l of microcrystalline cellulose at 90
g/liter in 50 mM sodium acetate pH 5.0, 0.01% TRITON.RTM. X-100, 20
.mu.l of culture supernatant, and 60 .mu.l of 50 mM sodium acetate
pH 5.0, 0.01% TRITON.RTM. X-100. The plates were sealed with tape.
The hydrolysis reaction was stopped by spinning the plate at 3500
rpm for 3 minutes. Then 12.5 .mu.l of the reaction supernatant were
added to 37.5 .mu.l MQ water in a 96 well PCR plate (Thermo Fisher
Scientific, Roskilde, Denmark). To this mixture 75 .mu.l of stop
solution was added. The stop solution was composed of 15 mg/ml
4-hydroxybenzhydrazide (Sigma Chemical Co., Inc., St. Louis, Mo.,
USA), 50 mg/ml K--Na-tartrate (Sigma Chemical Co., Inc., St. Louis,
Mo., USA) in 2% (w/v) NaOH. The plate was sealed with a lid and the
mixture was incubated at 95.degree. C. for 10 minutes and 5 minutes
at 20.degree. C. Then 100 .mu.l was transferred to a microtiter
plate and absorbance at 410 nm was measured using a SPECTRAMAX.RTM.
Plus 384 (Molecular Devices, Sunnyvale, Calif., USA). The
concentration of reducing sugar was proportional to the absorbance
at 410 nm of the oxidized 4-hydroxybenzhydrazide. The reducing
sugar content in the culture supernatants was measured by adding 1
.mu.l of culture supernatant to a mixture of 75 .mu.l of stop
solution and 49 .mu.l of milliQ water in a 96 well PCR plate. The
plate was sealed with a lid and the mixture was incubated at
95.degree. C. for 10 minutes and 5 minutes at 20.degree. C. Then
100 .mu.l was transferred to a microtiter plate and the absorbance
at 410 nm was measured. The absorbance at 410 nm from the cell
culture supernatant was subtracted from the absorbance at 410 nm of
the hydrolysis reaction, to measure the amount of reducing sugar
released by the enzymes.
[0555] Based on the level of hydrolysis of the microcrystalline
cellulose, one transformant expressing the A. fumigatus
cellobiohydrolase I variant AC1-870 was selected and designated A.
oryzae AC1-870.
[0556] For larger scale production, A. oryzae AC1-870 spores were
spread onto COVE sucrose slants and incubated for five days at
37.degree. C. The confluent spore slants were washed twice with 5
ml MQ water with 0.01% TWEEN.RTM. 20. The spore suspensions were
then used to inoculate a 500 ml flask containing 150 ml of G2-Gly
medium. The pre-culture was incubated at 30.degree. C. with
constant shaking at 200 rpm. After one day, the pre-culture was
used to inoculate four 500 ml flasks containing 200 ml of DAP-4C
medium. At day four post-inoculation, the culture broths were
collected by filtration through a bottle top MF75 Supor MachV 0.2
.mu.m PES filter.
Example 16: Site-Directed Mutagenesis of the Aspergillus fumigatus
Cellobiohydrolase I (AC1-846)
[0557] Plasmid pE621 (Example 3) was used to generate the A.
fumigatus cellobiohydrolase I AC1-846 variant (SEQ ID NO: 41 for
the mutant DNA sequence and SEQ ID NO: 42 for the variant), wherein
a TAC codon (Y475) was replaced with a TGG codon (475W).
[0558] Two synthetic primers for site-directed mutagenesis were
designed as shown below using a SOE primer design tool.
Site-directed mutagenesis of the synthetic gene encoding the
variant A. fumigatus cellobiohydrolase I AC1-621 was facilitated by
PCR amplifications of pE621 using the primers and procedure
described below:
TABLE-US-00006 Primer F-Y475W: (SEQ ID NO: 43)
5'-GTACAGGTGTGGCCCAGCACTGGGGACAGTGTGGCGGTATCGG-3' Primer R-Y475W:
(SEQ ID NO: 44) 5'-GTGCTGGGCCACACCTGTACCTCCAGGGTTG-3'
[0559] The mutation was introduced by PCR using a PHUSION.RTM.
High-Fidelity PCR Kit (New England Biolabs, MA, USA). The PCR
solution was composed of 10 .mu.l of 5.times.HF buffer (New England
Biolabs, MA, USA), 4 .mu.l of dNTPs (2.5 mM), 0.5 .mu.l of
PHUSION.RTM. High-Fidelity DNA polymerase (0.2 units/.mu.l) (New
England Biolabs, MA, USA), 0.25 .mu.l of primer F-Y475W (100
.mu.M), 0.25 .mu.l of primer R-Y475W (100 .mu.M), 5 .mu.l of
template DNA (pE621, 1 ng/.mu.l), and 30 .mu.l of deionized water
in a total volume of 50 .mu.l. he PCR was performed using an
Applied Biosystems.RTM. Veriti.RTM. 96 well thermal cycler
programmed for 1 cycle at 98.degree. C. for 30 seconds; and 19
cycles each at 98.degree. C. for 30 seconds, 55.degree. C. for 1
minute, and 72.degree. C. for 7 minutes. The PCR solution was then
held at 8.degree. C. until removed from the PCR machine.
[0560] Following the PCR, 10 units of Dpn I were added directly to
the PCR solution and incubated at 37.degree. C. for 1 hour. Then 1
.mu.l of the Dpn I treated PCR solution was transformed into ONE
SHOT.RTM. TOP10F' Chemically Competent E. coli cells according to
the manufacturer's protocol and spread onto LB plates supplemented
with 0.15 mg of ampicillin per ml. After incubation at 37.degree.
C. overnight, transformants were observed growing under selection
on the LB ampicillin plates. Four transformants were cultivated in
LB medium supplemented with 0.10 mg of ampicillin per ml and
plasmids were isolated using a QIAPREP.RTM. Spin Miniprep Kit.
[0561] The isolated plasmids were sequenced using an Applied
Biosystems 3730xl DNA Analyzer with primers F-pDau109 (SEQ ID NO:
29), R-pDau109 (SEQ ID NO: 30), F-pE596 (SEQ ID NO: 31) and R-pE596
(SEQ ID NO: 32), in order to determine a representative plasmid
that was free of PCR errors and contained the desired
mutations.
[0562] One plasmid clone free of PCR errors and containing the GTC
(V44) to ACT (44T) mutation and the TAC (Y475) to TGG (475W)
mutation was chosen and designated plasmid pE846.
Example 17: Site-Directed Mutagenesis of the Aspergillus fumigatus
Cellobiohydrolase I (AC1-860)
[0563] Plasmid pE846 (Example 16) was used to generate the A.
fumigatus cellobiohydrolase I AC1-860 variant (SEQ ID NO: 45 for
the mutant DNA sequence and SEQ ID NO: 46 for the variant), a TCC
codon (S265) was replaced with a CCT codon (265P).
[0564] Two synthetic primers for site-directed mutagenesis were
designed as shown below using a SOE primer design tool.
Site-directed mutagenesis of the synthetic gene encoding the
variant A. fumigatus cellobiohydrolase I AC1-846 was facilitated by
PCR amplifications of pE846 using the primers and procedure
described below:
TABLE-US-00007 Primer F-S265P: (SEQ ID NO: 35) 5'-CCCGA CGGCT GTGAC
TTCAA CCCTT TCAGG CAGGG CAACA AAACA TT-3' Primer R-S265P: (SEQ ID
NO: 36) 5'-GTTGA AGTCA CAGCC GTCGG GGTCA CACGT AC-3'
[0565] The mutation was introduced by PCR using a PHUSION.RTM.
High-Fidelity PCR Kit (New England Biolabs, MA, USA). The PCR
solution was composed of 10 .mu.l of 5.times.HF buffer (New England
Biolabs, MA, USA), 4 .mu.l of dNTPs (2.5 mM), 0.5 .mu.l of
PHUSION.RTM. High-Fidelity DNA polymerase (0.2 units/.mu.l) (New
England Biolabs, MA, USA), 0.25 .mu.l of primer F-S265P (100
.mu.M), 0.25 .mu.l of primer R-S265P (100 .mu.M), 5 .mu.l of
template DNA (pE846, 1 ng/.mu.l), and 30 .mu.l of deionized water
in a total volume of 50 .mu.l. he PCR was performed using an
Applied Biosystems.RTM. Veriti.RTM. 96 well thermal cycler
programmed for 1 cycle at 98.degree. C. for 30 seconds; and 19
cycles each at 98.degree. C. for 30 seconds, 55.degree. C. for 1
minute, and 72.degree. C. for 7 minutes. The PCR solution was then
held at 8.degree. C. until removed from the PCR machine. Following
the PCR, 10 units of Dpn I were added directly to the PCR solution
and incubated at 37.degree. C. for 1 hour. Then 1 .mu.l of the Dpn
I treated PCR solution was transformed into ONE SHOT.RTM. TOP10F'
Chemically Competent E. coli cells according to the manufacturer's
protocol and spread onto LB plates supplemented with 0.15 mg of
ampicillin per ml. After incubation at 37.degree. C. overnight,
transformants were observed growing under selection on the LB
ampicillin plates. Four transformants were cultivated in LB medium
supplemented with 0.10 mg of ampicillin per ml and plasmids were
isolated using a QIAPREP.RTM. Spin Miniprep Kit.
[0566] The isolated plasmids were sequenced using an Applied
Biosystems 3730xl DNA Analyzer with primers F-pDau109 (SEQ ID NO:
29), R-pDau109 (SEQ ID NO: 30), F-pE596 (SEQ ID NO: 31) and R-pE596
(SEQ ID NO: 32), in order to determine a representative plasmid
that was free of PCR errors and contained the desired
mutations.
[0567] One plasmid clone free of PCR errors and containing One
plasmid clone free of PCR errors and containing the GTC (V44) to
ACT (44T) mutation, the TAC (Y475) to TGG (475W) and the TCC (S265)
to CCT (265P) was chosen and designated plasmid pE860.
Example 18: Site-Directed Mutagenesis of the Aspergillus fumigatus
Cellobiohydrolase I (AC1-939)
[0568] Plasmid pE860 (Example 17) was used to generate the A.
fumigatus cellobiohydrolase I AC1-939 variant (SEQ ID NO: 47 for
the mutant DNA sequence and SEQ ID NO: 48 for the variant), an ACT
codon (T391) was replaced with a GAT codon (391D), a TCG codon
(S393) was replaced with a GAT codon (S393D) and a TCC codon (S394)
was replaced with a CCT codon (394P).
[0569] Two synthetic primers for site-directed mutagenesis were
designed as shown below using a SOE primer design tool.
Site-directed mutagenesis of the synthetic gene encoding the
variant A. fumigatus cellobiohydrolase I AC1-860 was facilitated by
PCR amplifications of pE860 using the primers and procedure
described below:
TABLE-US-00008 Primer F-T391D-S393D-S394P: (SEQ ID NO: 49) 5'-GTTGG
ATTCC AACTA CCCCA CCGAT GCCGA TCCTA CGACA CCGGG TGTCG CACG-3'
Primer R-T391D-S393D-S394P: (SEQ ID NO: 50) 5'-GGTGG GGTAG TTGGA
ATCCA ACCAG AGCAT GTTG-3'
[0570] The mutation was introduced by PCR using a PHUSION.RTM.
High-Fidelity PCR Kit (New England Biolabs, MA, USA). The PCR
solution was composed of 10 .mu.l of 5.times.HF buffer (New England
Biolabs, MA, USA), 4 .mu.l of dNTPs (2.5 mM), 0.5 .mu.l of
PHUSION.RTM. High-Fidelity DNA polymerase (0.2 units/.mu.l) (New
England Biolabs, MA, USA), 0.25 .mu.l of primer F-T391D-S393D-S394P
(100 .mu.M), 0.25 .mu.l of primer R-T391D-S393D-S394P (100 .mu.M),
5 .mu.l of template DNA (pE860, 1 ng/.mu.l), and 30 .mu.l of
deionized water in a total volume of 50 .mu.l. he PCR was performed
using an Applied Biosystems.RTM. Veriti.RTM. 96 well thermal cycler
programmed for 1 cycle at 98.degree. C. for 30 seconds; and 19
cycles each at 98.degree. C. for 30 seconds, 55.degree. C. for 1
minute, and 72.degree. C. for 7 minutes. The PCR solution was then
held at 8.degree. C. until removed from the PCR machine.
[0571] Following the PCR, 10 units of Dpn I were added directly to
the PCR solution and incubated at 37.degree. C. for 1 hour. Then 1
.mu.l of the Dpn I treated PCR solution was transformed into ONE
SHOT.RTM. TOP10F' Chemically Competent E. coli cells according to
the manufacturer's protocol and spread onto LB plates supplemented
with 0.15 mg of ampicillin per ml. After incubation at 37.degree.
C. overnight, transformants were observed growing under selection
on the LB ampicillin plates. Four transformants were cultivated in
LB medium supplemented with 0.10 mg of ampicillin per ml and
plasmids were isolated using a QIAPREP.RTM. Spin Miniprep Kit.
[0572] The isolated plasmids were sequenced using an Applied
Biosystems 3730xl DNA Analyzer with primers F-pDau109 (SEQ ID NO:
29), R-pDau109 (SEQ ID NO: 30), F-pE596 (SEQ ID NO: 31) and R-pE596
(SEQ ID NO: 32), in order to determine a representative plasmid
that was free of PCR errors and contained the desired
mutations.
[0573] One plasmid clone free of PCR errors and containing the GTC
(V44) to ACT (44T) mutation, the TAC (Y475) to TGG (475W), the TCC
(S265) to CCT (265P), the ACT (T391) to GAT (391D), the TCG (S393)
to GAT (393D) and the TCC (S394) to CCT (394P) mutations was chosen
and designated plasmid pE939.
Example 19: Expression of the Aspergillus fumigatus Variant
AC1-939
[0574] The expression plasmid pE939 (Example 18) was transformed
into Aspergillus oryzae MT3568 protoplasts according to Christensen
et al., 1988, supra and WO 2004/032648. A. oryzae MT3568
protoplasts were prepared according to the method of EP 0238023 B1,
pages 14-15.
[0575] Transformants were purified on COVE sucrose plates without
Triton X-100 through single conidia. Spores of the transformants
were inoculated into 96 deep well plates containing 0.50 ml of
DAP-4C medium and incubated stationary at 34.degree. C. for 6
days.
[0576] Production of the wild type A. fumigatus cellobiohydrolase I
AC1-939 by the transformants was analyzed from culture supernatants
of the 96 deep well cultivations. Expression was verified by
measuring released reducing sugars from hydrolysis of
microcrystalline cellulose. The hydrolysis was performed in 96 well
microtiter plates (NUNC Thermo Fisher Scientific, Roskilde,
Denmark) at 32.degree. C. and 1100 rpm. Each hydrolysis reaction
mixture contained 170 .mu.l of microcrystalline cellulose at 90
g/liter in 50 mM sodium acetate pH 5.0, 0.01% TRITON.RTM. X-100, 20
.mu.l of culture supernatant, and 60 .mu.l of 50 mM sodium acetate
pH 5.0, 0.01% TRITON.RTM. X-100. The plates were sealed with tape.
The hydrolysis reaction was stopped by spinning the plate at 3500
rpm for 3 minutes. Then 12.5 .mu.l of the reaction supernatant were
added to 37.5 .mu.l MQ water in a 96 well PCR plate (Thermo Fisher
Scientific, Roskilde, Denmark). To this mixture 75 .mu.l of stop
solution was added. The stop solution was composed of 15 mg/ml
4-hydroxybenzhydrazide (Sigma Chemical Co., Inc., St. Louis, Mo.,
USA), 50 mg/ml K--Na-tartrate (Sigma Chemical Co., Inc., St. Louis,
Mo., USA) in 2% (w/v) NaOH. The plate was sealed with a lid and the
mixture was incubated at 95.degree. C. for 10 minutes and 5 minutes
at 20.degree. C. Then 100 .mu.l was transferred to a microtiter
plate and absorbance at 410 nm was measured using a SPECTRAMAX.RTM.
Plus 384 (Molecular Devices, Sunnyvale, Calif., USA). The
concentration of reducing sugar was proportional to the absorbance
at 410 nm of the oxidized 4-hydroxybenzhydrazide. The reducing
sugar content in the culture supernatants was measured by adding 1
.mu.l of culture supernatant to a mixture of 75 .mu.l of stop
solution and 49 .mu.l of milliQ water in a 96 well PCR plate. The
plate was sealed with a lid and the mixture was incubated at
95.degree. C. for 10 minutes and 5 minutes at 20.degree. C. Then
100 .mu.l was transferred to a microtiter plate and the absorbance
at 410 nm was measured. The absorbance at 410 nm from the cell
culture supernatant was subtracted from the absorbance at 410 nm of
the hydrolysis reaction, to measure the amount of reducing sugar
released by the enzymes.
[0577] Based on the level of hydrolysis of the microcrystalline
cellulose one transformant expressing the wild type A. fumigatus
cellobiohydrolase I was selected and designated A. oryzae
AC1-939.
[0578] For larger scale production, A. oryzae AC1-939 spores were
spread onto COVE sucrose slants and incubated for five days at
37.degree. C. The confluent spore slants were washed twice with 5
ml MQ water with 0.01% TWEEN.RTM. 20. The spore suspensions were
then used to inoculate a 500 ml flask containing 150 ml of G2-Gly
medium. The pre-culture was incubated at 30.degree. C. with
constant shaking at 200 rpm. After one day, the pre-culture was
used to inoculate four 500 ml flasks containing 200 ml of DAP-4C
medium. At day four post-inoculation, the culture broths were
collected by filtration through a bottle top MF75 Supor MachV 0.2
.mu.m PES filter.
Example 20: Construction of an Aspergillus oryzae Expression Vector
Containing a Rasamsonia byssochiamydoides DNA Sequence Encoding
Cellobiohydrolase I
[0579] The genomic DNA sequence and deduced amino acid sequence of
the Rasamsonia byssochiamydoides (Talaromyces byssochiamydoides)
strain CBS413.71 GH7 cellobiohydrolase I gene is shown in SEQ ID
NO: 15 and SEQ ID NO: 16, respectively. The GH7 cellobiohydrolase I
gene SEQ ID NO: 15 is 1507 bp including the stop codon with two
predicted introns (604 to 667 and 1236 to 1310). Cloning of the R.
byssochiamydoides GH7 gene into pDau109 vector was conducted as
described in WO2012103300A2. The plasmid of pDau109 containing the
R. byssochiamydoides GH7 gene SEQ ID NO: 15 was designated
pE637.
Example 21: Construction of a Fusion Polypeptide of Rasamsonia
Byssochiamydoides Cellobiohydrolase I with Linker and Carbohydrate
Binding Module from Aspergillus fumigatus Cellobiohydrolase I
(RC1-638)
[0580] The codon-optimized synthetic gene encoding the Aspergillus
fumigatus cellobiohydrolase I is described in Example 2. The gene
encoding the R. byssochiamydoides cellobiohydrolase I is described
in Example 20.
[0581] To generate a gene encoding a fusion polypeptide of R.
byssochiamydoides cellobiohydrolase I with linker and carbohydrate
binding module (CBM) from A. fumigatus cellobiohydrolase I (SEQ ID
NOs: 51 and 52 for the fusion polypeptide DNA and amino acid
sequences, respectively), a DNA fragment encoding A. fumigatus
cellobiohydrolase I linker and CBM was assembled to the 3'-end of
the gene encoding the R. byssochlamydoides cellobiohydrolase I
using splicing overlap extension (50E) PCR.
[0582] Amplification of the DNA fragment encoding the A. fumigatus
cellobiohydrolase I linker and CBM was performed using a
PHUSION.RTM. High-Fidelity PCR Kit. The PCR solution was composed
of 10 .mu.l of 5.times.HF buffer, 4 .mu.l of dNTPs (2.5 mM), 0.5
.mu.l of PHUSION.RTM. DNA polymerase (0.2 units/.mu.l), 0.25 .mu.l
of primer F-50E638 (100 .mu.M), 0.25 .mu.l of primer R-50E638 (100
.mu.M), 10 .mu.l of template DNA (pE596 cellobiohydrolase I, 1
ng/.mu.l), and 25 .mu.l of deionized water in a total volume of 50
.mu.l. The PCR was performed using a GeneAmp.RTM. PCR System 9700
programmed for 1 cycle at 98.degree. C. for 30 seconds; and 30
cycles each at 98.degree. C. for 10 seconds, 55.degree. C. for 30
seconds, and 72.degree. C. for 1 minute. The PCR solution was then
held at 8.degree. C. until removed from the PCR machine.
TABLE-US-00009 Primer F-SOE638: (SEQ ID NO: 53)
5'-CAATCAACTCGACCTTCACCACTTCGGGCTCGAACCCTGGA GGCGGAACG-3' Primer
R-SOE638: (SEQ ID NO: 54)
5'-CTAGATCTCGAGTTACAAACACTGCGAGTAGTAG-3'
[0583] The PCR solution was submitted to 1% agarose gel
electrophoresis using TAE buffer where a 239 bp PCR fragment
encoding the A. fumigatus cellobiohydrolase I linker and CBM was
excised from the gel and purified using a MinElute Gel Extraction
Kit (QIAGEN Inc., Valencia, Calif., USA).
[0584] Amplification of the DNA fragment encoding the R.
byssochlamydoides wild-type cellobiohydrolase I was performed using
a PHUSION.RTM. High-Fidelity PCR Kit. The PCR solution was composed
of 10 .mu.l of 5.times.HF buffer, 4 .mu.l of dNTPs (2.5 mM), 0.5
.mu.l of PHUSION.RTM. DNA polymerase (0.2 units/.mu.l), 0.25 .mu.l
of primer F-pDAu109 (100 .mu.M), 0.25 .mu.l of primer R-50E637 (100
.mu.M), 10 .mu.l of template DNA (pE637--R. byssochlamydoides
cellobiohydrolase I, 1 ng/.mu.l), and 25 .mu.l of deionized water
in a total volume of 50 .mu.l. The PCR was performed using a
GeneAmp.RTM. PCR System 9700 programmed for 1 cycle at 98.degree.
C. for 30 seconds; and 30 cycles each at 98.degree. C. for 10
seconds, 55.degree. C. for 30 seconds, and 72.degree. C. for 1
minute. The PCR solution was then held at 8.degree. C. until
removed from the PCR machine.
TABLE-US-00010 Primer R-SOE637 (SEQ ID NO: 55)
5'-CGAAGTGGTGAAGGTCGAGTTGATTG-3'
[0585] The PCR solution was submitted to 1% agarose gel
electrophoresis using TAE buffer where a 1658 bp fragment encoding
the R. byssochlamydoides wild-type cellobiohydrolase I was excised
from the gel and purified using a MinElute Gel Extraction Kit.
[0586] The two purified DNA fragments were assembled using SOE PCR
and a PHUSION.RTM. High-Fidelity PCR Kit. The PCR solution was
composed of 10 .mu.l of 5.times.HF buffer, 4 .mu.l of dNTPs (2.5
mM), 0.5 .mu.l of PHUSION.RTM. DNA polymerase (0.2 units/.mu.l),
0.25 .mu.l of primer F-pDAu109 (100 .mu.M), 0.25 of R-pDAu109 (100
.mu.M), 2 .mu.l of gel purified fragment encoding A. fumigatus
cellobiohydrolase 1 linker and CBM, 2 .mu.l of DNA fragment
encoding R. byssochlamydoides cellobiohydrolase 1, and 31 .mu.l of
deionized water to give a final volume of 50 .mu.l. The PCR was
performed using a GeneAmp.RTM. PCR System 9700 programmed for 1
cycle at 98.degree. C. for 2 min; then 10 cycles of 98.degree. C.
for 20 seconds, 65.degree. C. for 20 seconds, and 72.degree. C. for
4 minutes; then followed by 20 cycles of 98.degree. C. for 20
seconds, 55.degree. C. for 20 seconds, and 72.degree. C. for 6
minutes. The PCR solution was then held at 6.degree. C. until
removed from the PCR machine.
[0587] The PCR generated DNA fragment was then digested with Bam HI
(New England Biolabs, Ipswich, Mass., USA) and XhoI (New England
Biolabs, Ipswich, Mass., USA) as follows. Twenty .mu.l of PCR
product were mixed with 2.3 .mu.l buffer 3.1 (New England Biolabs,
Ipswich, Mass., USA), 0.8 .mu.l of Bam HI, and 0.6 .mu.l of XhoI
and incubated at 37.degree. C. overnight. The resulting DNA product
was submitted to 1% agarose gel electrophoresis using TAE buffer. A
band of approximately 1717 bp was excised from the gel and purified
using a MinElute Gel Extraction Kit.
[0588] The 1717 bp fragment encoding the R. byssochlamydoides
cellobiohydrolase I with linker and carbohydrate binding module
(CBM) from A. fumigatus cellobiohydrolase I was cloned into pDAu109
digested with Bam HI and XhoI using T4 DNA ligase. The Bam HI-XhoI
digested pDau109 and the Bam HI/XhoI fragment containing the R.
byssochlamydoides cellobiohydrolase I with linker and carbohydrate
binding module (CBM) from A. fumigatus cellobiohydrolase I coding
sequence were mixed in a molar ratio of 1:3 (i.e., equal volumes of
gel purified products) and ligated with 50 units of T4 DNA ligase
in 1.times.T4 DNA ligase buffer with 1 mM ATP and incubated at
22.degree. C. for 10 minutes.
[0589] The ligation mixture was transformed into ONE SHOT.RTM.
TOP10F' Chemically Competent E. coli cells according to the
manufacturer's protocol and spread onto LB plates supplemented with
0.1 mg of ampicillin per ml. After incubation at 37.degree. C.
overnight, transformants were observed growing under selection on
the LB ampicillin plates. Two transformants were cultivated in LB
medium supplemented with 0.15 mg of ampicillin per ml and plasmids
were isolated using a QIAPREP.RTM. Spin Miniprep Kit.
[0590] The insertion of the DNA fragment encoding the R.
byssochlamydoides cellobiohydrolase I with linker and carbohydrate
binding module (CBM) from A. fumigatus cellobiohydrolase I into
pDAu109 was verified by sequencing. The isolated plasmids were
sequenced using an Applied Biosystems 3730xl DNA Analyzer with
vector primers F-pDau109 and R-pDau109 in order to determine a
representative plasmid that was free of PCR errors and contained
the correct insertion.
[0591] One plasmid clone free of PCR errors and containing the DNA
fragment encoding the R. byssochlamydoides cellobiohydrolase I with
linker and carbohydrate binding module (CBM) from A. fumigatus
cellobiohydrolase I was chosen and designated plasmid pE638. The
corresponding hybrid polypeptide was designated as RC1-638.
Example 22: Site-Directed Mutagenesis of the Fusion Polypeptide of
Rasamsonia Byssochlamydoides Cellobiohydrolase I with Linker and
Carbohydrate Binding Module from Aspergillus fumigatus
Cellobiohydrolase I (RC1-899)
[0592] Plasmid pE638 (Example 21) was used to generate R.
byssochlamydoides-A. fumigatus fusion cellobiohydrolase I variant
(RC1-899). For the RC1-899 the SEQ ID NO: 56 for the mutant DNA
sequence and SEQ ID NO: 57 for the variant a TAC codon (Y499) was
replaced with a TGG codon (499W).
[0593] Two synthetic primers for site-directed mutagenesis were
designed using a SOE primer design tool. Site-directed mutageneis
of the fusion gene endcoding R. byssochlamydoides cellobiohydrolase
I with linker and carbohydrate binding module (CBM) from A.
fumigatus cellobiohydrolase I was facilitated by PCR amplification
of pE638 using primers F-Y527W and R-Y527W and the procedure
described below.
TABLE-US-00011 Primer F-Y527W: (SEQ ID NO: 58)
5'-ATACCTGTCAGAAATTGAACGACTGGTACTCGCAGTGT TTGTAAGCTTC-3' Primer
R-Y527W: (SEQ ID NO: 59) 5'-GTCGTTCAATTTCTGACAGGTATAAGGCGATG-3'
Primer F-pE638: (SEQ ID NO: 60) 5'-CCTCAGCCGAACTCCGACATTGC-3'
Primer R-pE638: (SEQ ID NO: 61) 5'-GCAATGTCGGAGTTCGGCTGAGG-3'
[0594] The mutation was introduced by PCR using a PHUSION.RTM.
High-Fidelity PCR Kit (New England Biolabs Inc. MA, USA). The PCR
solutions were composed of 10 .mu.l of 5.times.HF buffer, 4 .mu.l
of dNTPs (2.5 mM), 0.5 .mu.l of PHUSION.RTM. DNA polymerase (0.2
units/.mu.l), 0.25 .mu.l of primer F-Y527W (100 .mu.M), 0.25 .mu.l
of primer R-Y527W (100 .mu.M), 5 .mu.l of template DNA (pE638, 1
ng/.mu.l), and 30 .mu.l of deionized water in a total volume of 50
.mu.l. he PCR was performed using an Applied Biosystems.RTM.
Veriti.RTM. 96 well thermal cycler programmed for 1 cycle at
98.degree. C. for 30 seconds; and 19 cycles each at 98.degree. C.
for 30 seconds, 55.degree. C. for 1 minute, and 72.degree. C. for 7
minutes. The PCR solution was then held at 8.degree. C. until
removed from the PCR machine.
[0595] Following the PCR, 10 units of Dpn I were added directly to
the PCR solution and incubated at 37.degree. C. for 1 hour. Then 1
.mu.l of the Dpn I treated PCR solution was transformed into ONE
SHOT.RTM. TOP10F' Chemically Competent E. coli cells according to
the manufacturer's protocol and spread onto LB plates supplemented
with 0.15 mg of ampicillin per ml. After incubation at 37.degree.
C. overnight, transformants were observed growing under selection
on the LB ampicillin plates. Four transformants were cultivated in
LB medium supplemented with 0.10 mg of ampicillin per ml and
plasmids were isolated using a QIAPREP.RTM. Spin Miniprep Kit
(QIAGEN Inc., Valencia, Calif., USA).
[0596] The isolated mutant plasmids of pE638 were sequenced using
an Applied Biosystems 3730xl DNA Analyzer (Applied Biosystems,
Foster City, Calif., USA) with primers F-pDau109 (SEQ ID NO: 29),
R-pDau109 (SEQ ID NO: 30), F-pE638 (SEQ ID NO: 60) and R-pE638 (SEQ
ID NO: 61), in order to determine a representative plasmid that was
free of PCR errors and contained the desired mutations.
[0597] One plasmid clone free of PCR errors and containing the TAC
(Y499) to TGG (499W) mutation was chosen and designated plasmid
pE899 and the corresponding polypeptide was designated as
RC1-899.
Example 23: Site-Directed Mutagenesis of the Fusion Polypeptide of
Rasamsonia Byssochlamydoides Cellobiohydrolase I with Linker and
Carbohydrate Binding Module from Aspergillus fumigatus
Cellobiohydrolase I (RC1-1127)
[0598] Plasmid pE899 (Example 22) was used to generate R.
byssochlamydoides-A. fumigatus fusion cellobiohydrolase I variant
RC1-1127 (SEQ ID NO: 62 for the mutant DNA sequence and SEQ ID NO:
63 for the variant), wherein an AAT codon (N388) was replaced with
a GAC codon (388D), a TCT codon (S390) was replaced with a GAC
codon (S390D) and a GCT codon (A391) was replaced with a CCT codon
(391P). The N388D, S390D, and A391P substitutions of SEQ ID NO: 63
correspond to positions 391, 393, and 394 of SEQ ID NO: 4,
respectively.
[0599] Two synthetic primers for site-directed mutagenesis were
designed using a SOE primer design tool. Site-directed mutagenesis
of the gene encoding the RC1-899 variant of the fusion polypeptide
of Rasamsonia byssochlamydoides cellobiohydrolase I with linker and
carbohydrate binding module from Aspergillus fumigatus
cellobiohydrolase I was facilitated by PCR amplifications of pE899
using the primers and procedure described below:
TABLE-US-00012 Primer F-N388D S390D A391P: (SEQ ID NO: 64)
5'-GGTTGGACAGCATTTATCCAACAGACGCAGACCCTAGCACT CCTGGTGCTGCTCG-3'
Primer R-N388D S390D A391P: (SEQ ID NO: 65)
5'-TGTTGGATAAATGCTGTCCAACCACAGCATGTTTG-3'
[0600] The mutation was introduced by PCR using a PHUSION.RTM.
High-Fidelity PCR Kit (Finnzymes Oy, Espoo, Finland). The PCR
solution was composed of 10 .mu.l of 5.times.HF buffer (Finnzymes
Oy, Espoo, Finland), 4 .mu.l of dNTPs (2.5 mM), 0.5 .mu.l of
PHUSION.RTM. DNA polymerase (0.2 units/.mu.l) (Finnzymes Oy, Espoo,
Finland), 0.25 .mu.l of primer F-N388D S390D A391P (100 .mu.M),
0.25 .mu.l of primer R-N388D S390D A391P (100 .mu.M), 5 .mu.l of
template DNA (pE899, 1 ng/.mu.l), and 30 .mu.l of deionized water
in a total volume of 50 .mu.l. he PCR was performed using an
Applied Biosystems.RTM. Veriti.RTM. 96 well thermal cycler
programmed for 1 cycle at 98.degree. C. for 30 seconds; and 19
cycles each at 98.degree. C. for 30 seconds, 55.degree. C. for 1
minute, and 72.degree. C. for 7 minutes. The PCR solution was then
held at 8.degree. C. until removed from the PCR machine.
[0601] Following the PCR, 10 units of Dpn I were added directly to
the PCR solution and incubated at 37.degree. C. for 1 hour. Then 1
.mu.l of the Dpn I treated PCR solution was transformed into ONE
SHOT.RTM. TOP10F' Chemically Competent E. coli cells according to
the manufacturer's protocol and spread onto LB plates supplemented
with 0.15 mg of ampicillin per ml. After incubation at 37.degree.
C. overnight, transformants were observed growing under selection
on the LB ampicillin plates. Four transformants were cultivated in
LB medium supplemented with 0.10 mg of ampicillin per ml and
plasmids were isolated using a QIAPREP.RTM. Spin Miniprep Kit
(QIAGEN Inc., Valencia, Calif., USA).
[0602] The isolated plasmids were sequenced using an Applied
Biosystems 3730xl DNA Analyzer (Applied Biosystems, Foster City,
Calif., USA) with primers F-pDau109 (SEQ ID NO: 29), R-pDau109 (SEQ
ID NO: 30), F-pE596 (SEQ ID NO: 31) and R-pE596 (SEQ ID NO: 32), in
order to determine a representative plasmid that was free of PCR
errors and contained the desired mutations.
[0603] One plasmid clone free of PCR errors and containing the AAT
codon (N388) to GAC codon (388D) mutation (corresponding to
position 391 of SEQ ID NO: 4), the TCT codon (S390) to GAC (390D)
mutation (corresponding to position 393 of SEQ ID NO: 4) and GCT
(A391) to CCT codon (391P) mutation (corresponding to position 394
of SEQ ID NO: 4) was chosen and designated plasmid pE1127.
Example 24: Expression of the A. fumigatus Cellobiohydrolase I
Variants AC1-846, AC1-860 and R. Byssochlamydoides-A. fumigatus
Fusion Protein Variants RC1-899 and RC1-1127
[0604] The expression plasmids pE846 (Example 16), pE860 (Example
17), pE899 (Example 22), and pE1127 (Example 23) and were
transformed into Aspergillus oryzae MT3568 protoplasts according to
Christensen et al., 1988, supra and WO 2004/032648. A. oryzae
MT3568 protoplasts were prepared according to the method of EP
0238023 B1, pages 14-15.
[0605] Transformants were purified on COVE sucrose plates without
Triton X-100 through single conidia. Spores of the transformants
were inoculated into 96 deep well plates containing 0.50 ml of
DAP-4C medium and incubated stationary at 34.degree. C. for 6
days.
[0606] Production of the A. fumigatus cellobiohydrolase variants
AC1-846 and AC1-860, and R. byssochlamydoides-A. fumigatus fusion
cellobiohydrolase I variants RC1-899 and RC1-1127 by the
transformants were analyzed from culture supernatants of the 96
deep well cultivations. Expression was verified by measuring
released reducing sugars from hydrolysis of microcrystalline
cellulose. The hydrolysis was performed in 96 well microtiter
plates (NUNC Thermo Fisher Scientific, Roskilde, Denmark) at
32.degree. C. and 1100 rpm. Each hydrolysis reaction mixture
contained 170 .mu.l of microcrystalline cellulose at 90 g/liter in
50 mM sodium acetate pH 5.0, 0.01% TRITON.RTM. X-100, 20 .mu.l of
culture supernatant, and 60 .mu.l of 50 mM sodium acetate pH 5.0,
0.01% TRITON.RTM. X-100. The plates were sealed with tape. The
hydrolysis reaction was stopped by spinning the plate at 3500 rpm
for 3 minutes. Then 12.5 .mu.l of the reaction supernatant were
added to 37.5 .mu.l MQ water in a 96 well PCR plate (Thermo Fisher
Scientific, Roskilde, Denmark). To this mixture 75 .mu.l of stop
solution was added. The stop solution was composed of 15 mg/ml
4-hydroxybenzhydrazide (Sigma Chemical Co., Inc., St. Louis, Mo.,
USA), 50 mg/ml K--Na-tartrate (Sigma Chemical Co., Inc., St. Louis,
Mo., USA) in 2% (w/v) NaOH. The plate was sealed with a lid and the
mixture was incubated at 95.degree. C. for 10 minutes and 5 minutes
at 20.degree. C. Then 100 .mu.l was transferred to a microtiter
plate and absorbance at 410 nm was measured using a SPECTRAMAX.RTM.
Plus 384 (Molecular Devices, Sunnyvale, Calif., USA). The
concentration of reducing sugar was proportional to the absorbance
at 410 nm of the oxidized 4-hydroxybenzhydrazide. The reducing
sugar content in the culture supernatants was measured by adding 1
.mu.l of culture supernatant to a mixture of 75 .mu.l of stop
solution and 49 .mu.l of milliQ water in a 96 well PCR plate. The
plate was sealed with a lid and the mixture was incubated at
95.degree. C. for 10 minutes and 5 minutes at 20.degree. C. Then
100 .mu.l was transferred to a microtiter plate and the absorbance
at 410 nm was measured. The absorbance at 410 nm from the cell
culture supernatant was subtracted from the absorbance at 410 nm of
the hydrolysis reaction, to measure the amount of reducing sugar
released by the enzymes.
[0607] Based on the level of hydrolysis of the microcrystalline
cellulose, one transformant expressing the A. fumigatus
cellobiohydrolase I variant AC1-846 was selected and designated A.
oryzae AC1-846, one transformant expressing the A. fumigatus
cellobiohydrolase I variant AC1-860 was selected and designated A.
oryzae AC1-860, one transformant expressing the R.
byssochlamydoides-A. fumigatus fusion cellobiohydrolase I variant
RC1-899 was selected and designated A. oryzae RC1-899, and one
transformant expressing the R. byssochlamydoides-A. fumigatus
fusion cellobiohydrolase I variant RC1-1127 was selected and
designated A. oryzae RC1-1127.
[0608] For larger scale production, A. oryzae AC1-846, A. oryzae
AC1-860, A. oryzae RC1-899 or A. oryzae RC1-1127 spores were spread
onto COVE sucrose slants and incubated for five days at 37.degree.
C. The confluent spore slants were washed twice with 5 ml MQ water
with 0.01% TWEEN.RTM. 20. The spore suspensions were then used to
inoculate a 500 ml flask containing 150 ml of G2-Gly medium. The
pre-culture was incubated at 30.degree. C. with constant shaking at
200 rpm. After one day, the pre-culture was used to inoculate four
500 ml flasks containing 200 ml of DAP-4C medium. At day four
post-inoculation, the culture broths were collected by filtration
through a bottle top MF75 Supor MachV 0.2 .mu.m PES filter.
Example 25: Purification of the Aspergillus fumigatus Variants
AC1-846, AC1-860, AC1-870 and of the R. byssochiamydoides-A.
fumigatus Fusion Cellobiohydrolase I Variants RC1-899 and
RC1-1127
[0609] The broths of A. oryzae AC1-846, A. oryzae AC1-860, A.
oryzae AC1-870, A. oryzae RC1-899 or A. oryzae RC1-1127 were
filtered using a 0.22 .mu.m PES filter (Nalge Nunc International
Corp., Rochester, N.Y., USA), followed by addition of ammonium
sulphate to a concentration of 1.8 M and another filtration.
[0610] Each filtrate was purified according to the following
procedure. The filtrate was loaded onto a Phenyl SEPHAROSE.RTM. 6
Fast Flow column (high sub) (GE Healthcare, Piscataway, N.J., USA)
equilibrated with 1.8 M ammonium sulphate, 25 mM HEPES pH 7.0.
After a wash with 2 CV 1.8 M ammonium sulphate followed by 1 CV
0.54 M ammonium sulphate, the bound proteins were batch eluted with
25 mM HEPES pH 7.0.
[0611] The elution of the protein was monitored at 280 nm and
fractions were collected and analyzed by SDS-PAGE using 12-well
NUPAGE.RTM. 4-12% Bis-Tris gel (GE Healthcare, Piscataway, N.J.,
USA). The fractions were pooled based on SDS-PAGE as above and
applied to a SEPHADEX.TM. G-25 (medium) column (GE Healthcare,
Piscataway, N.J., USA) equilibrated with 25 mM MES pH 6.0.
Fractions were collected, analyzed by SDS-PAGE as above, and
pooled.
[0612] The pooled fractions were applied to a 53 ml RESOURCE.TM.
15Q column (GE Healthcare, Piscataway, N.J., USA) equilibrated with
25 mM MES pH 6.0 and bound proteins were eluted with a linear
100-200 mM sodium chloride gradient for 2 CV followed by 1 CV 1 M
sodium chloride. The elution of the protein was monitored at 280 nm
and fractions with high absorbance at 280 nm were analyzed on
SDS-PAGE. Fractions with high content of cellobiohydrolase I were
pooled.
Example 26: Site-Directed Mutagenesis of the Aspergillus fumigatus
Cellobiohydrolase I (AC1-714, AC1-859, AC1-940, AC1-942, AC1-943,
AC1-946, AC1-959, AC1-964, AC1-971, AC1-974)
[0613] The A. fumigatus cellobiohydrolase I variants AC1-714,
AC1-859, AC1-940, AC1-942, AC1-943, AC1-946, AC1-959, AC1-964,
AC1-971, AC1-974 (containing the alterations and sequences shown in
Table 1) were prepared in a similar manner to that described in
Examples 3, 5, 14, and 16-17; expressed in a similar manner to that
described in Examples 4, 6, 15, and 19; and purified in a similar
manner to that described in Examples 7 and 25.
TABLE-US-00013 TABLE 1 Polypeptide Name Polypeptide ID Alterations
AC1-596 SEQ ID NO: 4 N/A (wild-type) AC1-621 SEQ ID NO: 26 V44T
AC1-625 SEQ ID NO: 28 S265P AC1-714 SEQ ID NO: 67 G4C + A72C
AC1-846 SEQ ID NO: 42 V44T + Y475W AC1-859 SEQ ID NO: 69 V44T +
S265P AC1-860 SEQ ID NO: 46 V44T + S265P + Y475W AC1-870 SEQ ID NO:
38 T391D + S393D + S394P AC1-939 SEQ ID NO: 48 V44T + S265P + T391D
+ S393D + S394P + Y475W AC1-940 SEQ ID NO: 71 S393D + S394P AC1-942
SEQ ID NO: 81 T391W + S393D + S394P AC1-943 SEQ ID NO: 83 T391V +
S393D + S394P AC1-946 SEQ ID NO: 85 T391N + S393D + S394P AC1-959
SEQ ID NO: 73 V44I AC1-964 SEQ ID NO: 75 V44M AC1-971 SEQ ID NO: 77
V44N AC1-974 SEQ ID NO: 79 V44K RC1-638 SEQ ID NO: 52 N/A (fusion)
RC1-899 SEQ ID NO: 57 Y499W (corresponding to position 501 of SEQ
ID NO: 4) RC1-1127 SEQ ID NO: 63 N388D + S390D + A391P + Y499W
(corresponding to positions 391, 393, 394 and 501 of SEQ ID NO: 4,
respectively)
Example 27: Determination of Td by Differential Scanning
Calorimetry of the A. fumigatus Cellobiohydrolase I AC1-596
Wild-Type Polypeptide; A. fumigatus Cellobiohydrolase I Variants
AC1-870, AC1-939, AC1-940, AC1-942, AC1-943, and AC1-946; and R.
byssochiamydoides Cellobiohydrolase I Fusion Variant RC1-1127
[0614] The thermostability of the Aspergillus fumigatus wild-type
cellobiohydrolase I AC1-596; the cellobiohydrolase I variants
AC1-870, AC1-939, AC1-940, AC1-942, AC1-943, and AC1-946; and the
R. byssochiamydoides cellobiohydrolase I fusion variant RC1-1127
were determined by Differential Scanning calorimetry (DSC) as
described in Example 13.
[0615] The results demonstrated that the Aspergillus fumigatus
cellobiohydrolase I AC1-870 variant had 4.degree. C. stabilization
under the tested assay conditions compared to the wild-type
Aspergillus fumigatus cellobiohydrolase I AC1-596; the Aspergillus
fumigatus variant AC1-939 had a had 7.degree. C. stabilization
under the tested assay conditions compared to the wild-type
Aspergillus fumigatus cellobiohydrolase I AC1-596; the Aspergillus
fumigatus variant AC1-940 had a had 4.degree. C. stabilization
under the tested assay conditions compared to the wild-type
Aspergillus fumigatus cellobiohydrolase I AC1-596, the Aspergillus
fumigatus variant AC1-942 had a had 5.degree. C. stabilization
under the tested assay conditions compared to the wild-type
Aspergillus fumigatus cellobiohydrolase I AC1-596, the Aspergillus
fumigatus variant AC1-943 had a had 2.degree. C. stabilization
under the tested assay conditions compared to the wild-type
Aspergillus fumigatus cellobiohydrolase I AC1-596, the Aspergillus
fumigatus variant AC1-946 had a had 4.degree. C. stabilization
under the tested assay conditions compared to the wild-type
Aspergillus fumigatus cellobiohydrolase I AC1-596, and the R.
byssochiamydoides cellobiohydrolase I fusion variant RC1-1127 had
2.degree. C. stabilization under the tested assay conditions
compared to the corresponding cellobiohydrolase I parent
RC1-899.
Example 28: Comparison of the Effect of Aspergillus fumigatus
Cellobiohydrolase I Variants AC1-621, AC1-625, AC1-859, AC1-860,
AC1-870, AC1-939, AC1-940, and AC1-942 Against Aspergillus
fumigatus Cellobiohydrolase I AC1-596 Wild-Type Polypeptide on the
Hydrolysis of Unwashed PCS by a Cellulase Enzyme Composition
[0616] The Aspergillus fumigatus cellobiohydrolase I variants
AC1-621 (Example 7), AC1-625 (Example 7), AC1-859 (Example 26),
AC1-860 (Example 25), AC1-870 (Example 25), AC1-939 (Example 19),
AC1-940 (Example 26) and AC1-942 (Example 26) were added
individually to the cellulolytic enzyme composition without
cellobiohydrolase I (Example 10) at 50.degree. C., 55.degree. C.
and 60.degree. C. using unwashed PCS as a substrate, and compared
against the Aspergillus fumigatus cellobiohydrolase I AC1-596
wild-type polypeptide added to the cellulolytic enzyme composition
without cellobiohydrolase I. Each cellobiohydrolase I was added
individually at 3.0 mg enzyme protein per g cellulose to 5.108 mg
enzyme protein of the cellulase enzyme composition without
cellobiohydrolase I per g cellulose.
[0617] The assay was performed as described in Example 9. The
reactions with unwashed PCS (20% total solids) were conducted for
72 hours at 50.degree. C., 55.degree. C. and 60.degree. C. in 100
mM sodium acetate pH 5.0 buffer containing 1 mM manganese sulfate.
All reactions were performed in quadruplicate and shaking at 200
rpm throughout the hydrolysis.
[0618] The results for experiments at 50.degree. C., 55.degree. C.
and 60.degree. C. are shown in FIG. 7. The results demonstrated
that the cellulase enzyme composition containing the Aspergillus
fumigatus cellobiohydrolase I AC1-621 variant polypeptide had
significantly higher cellulose conversion than the cellulase enzyme
composition that included Aspergillus fumigatus cellobiohydrolase I
AC1-596 wild-type polypeptide at 50.degree. C. In addition, the
results demonstrated that the cellulase enzyme composition
containing the Aspergillus fumigatus cellobiohydrolase I AC1-625
variant polypeptide, the cellulase enzyme composition containing
the Aspergillus fumigatus cellobiohydrolase I AC1-859 variant
polypeptide, the cellulase enzyme composition containing the
Aspergillus fumigatus cellobiohydrolase I AC1-860 variant
polypeptide, the cellulase enzyme composition containing the
Aspergillus fumigatus cellobiohydrolase I AC1-870 variant
polypeptide, the cellulase enzyme composition containing the
Aspergillus fumigatus cellobiohydrolase I AC1-939 variant
polypeptide, and the cellulase enzyme composition containing the
Aspergillus fumigatus cellobiohydrolase I AC1-940 variant
polypeptide had significantly higher cellulose conversion than the
cellulase enzyme composition that included Aspergillus fumigatus
cellobiohydrolase I AC1-596 wild-type polypeptide at 50.degree. C.,
55.degree. C. and 60.degree. C. Lastly, the data demonstrated that
the cellulase enzyme composition containing the Aspergillus
fumigatus cellobiohydrolase I AC1-942 variant polypeptide had
significantly higher cellulose conversion than the cellulase enzyme
composition that included Aspergillus fumigatus cellobiohydrolase I
AC1-596 wild-type polypeptide at 55.degree. C. and 60.degree.
C.
Example 29: Comparison of the Effect of Aspergillus fumigatus
Cellobiohydrolase I AC1-621, AC1-714, AC1-959, AC1-964, AC1-971,
and AC1-974 Against Aspergillus fumigatus Cellobiohydrolase I
AC1-596 Wild-Type Polypeptide on the Hydrolysis of Unwashed PCS by
a Cellulase Enzyme Composition
[0619] The Aspergillus fumigatus cellobiohydrolase I variants
AC1-621 (Example 7), AC1-714 (Example 26), AC1-959 (Example 26),
AC1-964 (Example 26), AC1-971 (Example 26), and AC1-974 variant
polypeptide (Example 26) were added to the cellulolytic enzyme
composition without cellobiohydrolase I (Example 10) at 50.degree.
C., 55.degree. C. and 60.degree. C. using unwashed PCS as a
substrate, and compared against the Aspergillus fumigatus
cellobiohydrolase I AC1-596 wild-type polypeptide added to the
cellulolytic enzyme composition without cellobiohydrolase I. Each
cellobiohydrolase I was added individually at 3.0 mg enzyme protein
per g cellulose to 5.108 mg enzyme protein of the cellulase enzyme
composition without cellobiohydrolase I per g cellulose.
[0620] The assay was performed as described in Example 9. The
reactions with unwashed PCS (20% total solids) were conducted for
72 hours at 50.degree. C., 55.degree. C. and 60.degree. C. in 100
mM sodium acetate pH 5.0 buffer containing 1 mM manganese sulfate.
All reactions were performed in quadruplicate and shaking at 200
rpm throughout the hydrolysis.
[0621] As shown in FIG. 8, the results demonstrated that the
cellulase enzyme composition containing the Aspergillus fumigatus
cellobiohydrolase I AC1-714 variant polypeptide had significantly
higher cellulose conversion than the cellulase enzyme composition
that included Aspergillus fumigatus cellobiohydrolase I AC1-596
wild-type polypeptide at 50.degree. C., 55.degree. C. and
60.degree. C. As shown in FIG. 9, the results demonstrated that the
cellulase enzyme composition containing the Aspergillus fumigatus
cellobiohydrolase I AC1-621 variant polypeptide, the cellulase
enzyme composition containing the Aspergillus fumigatus
cellobiohydrolase I AC1-959 variant polypeptide, the cellulase
enzyme composition containing the Aspergillus fumigatus
cellobiohydrolase I AC1-964 variant polypeptide, the cellulase
enzyme composition containing the Aspergillus fumigatus
cellobiohydrolase I AC1-971 variant polypeptide, and the cellulase
enzyme composition containing the Aspergillus fumigatus
cellobiohydrolase I AC1-974 variant polypeptide had significantly
higher cellulose conversion than the cellulase enzyme composition
that included Aspergillus fumigatus cellobiohydrolase I AC1-596
wild-type polypeptide at 50.degree. C. In addition, the results
demonstrated that the cellulase enzyme composition containing the
Aspergillus fumigatus cellobiohydrolase I AC1-974 variant
polypeptide had significantly higher cellulose conversion than the
cellulase enzyme composition that included Aspergillus fumigatus
cellobiohydrolase I AC1-596 wild-type polypeptide at 55.degree.
C.
[0622] The invention described and claimed herein is not to be
limited in scope by the specific aspects herein disclosed, since
these aspects are intended as illustrations of several aspects of
the invention. Any equivalent aspects are intended to be within the
scope of this invention. Indeed, various modifications of the
invention in addition to those shown and described herein will
become apparent to those skilled in the art from the foregoing
description. Such modifications are also intended to fall within
the scope of the appended claims. In the case of conflict, the
present disclosure including definitions will control.
Sequence CWU 1
1
8511599DNAAspergillus fumigatus 1atgctggcct ccaccttctc ctaccgcatg
tacaagaccg cgctcatcct ggccgccctt 60ctgggctctg gccaggctca gcaggtcggt
acttcccagg cggaagtgca tccgtccatg 120acctggcaga gctgcacggc
tggcggcagc tgcaccacca acaacggcaa ggtggtcatc 180gacgcgaact
ggcgttgggt gcacaaagtc ggcgactaca ccaactgcta caccggcaac
240acctgggaca cgactatctg ccctgacgat gcgacctgcg catccaactg
cgcccttgag 300ggtgccaact acgaatccac ctatggtgtg accgccagcg
gcaattccct ccgcctcaac 360ttcgtcacca ccagccagca gaagaacatt
ggctcgcgtc tgtacatgat gaaggacgac 420tcgacctacg agatgtttaa
gctgctgaac caggagttca ccttcgatgt cgatgtctcc 480aacctcccct
gcggtctcaa cggtgctctg tactttgtcg ccatggacgc cgacggtggc
540atgtccaagt acccaaccaa caaggccggt gccaagtacg gtactggata
ctgtgactcg 600cagtgccctc gcgacctcaa gttcatcaac ggtcaggcca
acgttgaagg gtggcagccc 660tcctccaacg atgccaatgc gggtaccggc
aaccacgggt cctgctgcgc ggagatggat 720atctgggagg ccaacagcat
ctccacggcc ttcacccccc atccgtgcga cacgcccggc 780caggtgatgt
gcaccggtga tgcctgcggt ggcacctaca gctccgaccg ctacggcggc
840acctgcgacc ccgacggatg tgatttcaac tccttccgcc agggcaacaa
gaccttctac 900ggccctggca tgaccgtcga caccaagagc aagtttaccg
tcgtcaccca gttcatcacc 960gacgacggca cctccagcgg caccctcaag
gagatcaagc gcttctacgt gcagaacggc 1020aaggtgatcc ccaactcgga
gtcgacctgg accggcgtca gcggcaactc catcaccacc 1080gagtactgca
ccgcccagaa aagcctgttc caggaccaga acgtcttcga aaagcacggc
1140ggcctcgagg gcatgggtgc tgccctcgcc cagggcatgg ttctcgtcat
gtccctgtgg 1200gatgatcact cggccaacat gctctggctc gacagcaact
acccgaccac tgcctcttcc 1260accactcccg gcgtcgcccg tggtacctgc
gacatctcct ccggcgtccc tgcggatgtc 1320gaggcgaacc accccgacgc
ctacgtcgtc tactccaaca tcaaggtcgg ccccatcggc 1380tcgaccttca
acagcggtgg ctcgaacccc ggtggcggaa ccaccacgac aactaccacc
1440cagcctacta ccaccacgac cacggctgga aaccctggcg gcaccggagt
cgcacagcac 1500tatggccagt gtggtggaat cggatggacc ggacccacaa
cctgtgccag cccttatacc 1560tgccagaagc tgaatgatta ttactctcag
tgcctgtag 159921599DNAAspergillus fumigatus 2atgctggcct ccaccttctc
ctaccgcatg tacaagaccg cgctcatcct ggccgccctt 60ctgggctctg gccaggctca
gcaggtcggt acttcccagg cggaagtgca tccgtccatg 120acctggcaga
gctgcacggc tggcggcagc tgcaccacca acaacggcaa ggtggtcatc
180gacgcgaact ggcgttgggt gcacaaagtc ggcgactaca ccaactgcta
caccggcaac 240acctgggaca cgactatctg ccctgacgat gcgacctgcg
catccaactg cgcccttgag 300ggtgccaact acgaatccac ctatggtgtg
accgccagcg gcaattccct ccgcctcaac 360ttcgtcacca ccagccagca
gaagaacatt ggctcgcgtc tgtacatgat gaaggacgac 420tcgacctacg
agatgtttaa gctgctgaac caggagttca ccttcgatgt cgatgtctcc
480aacctcccct gcggtctcaa cggtgctctg tactttgtcg ccatggacgc
cgacggtggc 540atgtccaagt acccaaccaa caaggccggt gccaagtacg
gtactggata ctgtgactcg 600cagtgccctc gcgacctcaa gttcatcaac
ggtcaggcca acgttgaagg gtggcagccc 660tcctccaacg atgccaatgc
gggtaccggc aaccacgggt cctgctgcgc ggagatggat 720atctgggagg
ccaacagcat ctccacggcc ttcacccccc atccgtgcga cacgcccggc
780caggtgatgt gcaccggtga tgcctgcggt ggcacctaca gctccgaccg
ctacggcggc 840acctgcgacc ccgacggatg tgatttcaac tccttccgcc
agggcaacaa gaccttctac 900ggccctggca tgaccgtcga caccaagagc
aagtttaccg tcgtcaccca gttcatcacc 960gacgacggca cctccagcgg
caccctcaag gagatcaagc gcttctacgt gcagaacggc 1020aaggtgatcc
ccaactcgga gtcgacctgg accggcgtca gcggcaactc catcaccacc
1080gagtactgca ccgcccagaa aagcctgttc caggaccaga acgtcttcga
aaagcacggc 1140ggcctcgagg gcatgggtgc tgccctcgcc cagggcatgg
ttctcgtcat gtccctgtgg 1200gatgatcact cggccaacat gctctggctc
gacagcaact acccgaccac tgcctcttcc 1260accactcccg gcgtcgcccg
tggtacctgc gacatctcct ccggcgtccc tgcggatgtc 1320gaggcgaacc
accccgacgc ctacgtcgtc tactccaaca tcaaggtcgg ccccatcggc
1380tcgaccttca acagcggtgg ctcgaacccc ggtggcggaa ccaccacgac
aactaccacc 1440cagcctacta ccaccacgac cacggctgga aaccctggcg
gcaccggagt cgcacagcac 1500tatggccagt gtggtggaat cggatggacc
ggacccacaa cctgtgccag cccttatacc 1560tgccagaagc tgaatgatta
ttactctcag tgcctgtag 159931599DNAAspergillus
fumigatussig_peptide(1)..(26)CDS(1)..(1596)mat_peptide(79)..(1596)
3atg ttg gcc tcc acg ttc tcc tat cgc atg tac aaa aca gcg ctc atc
48Met Leu Ala Ser Thr Phe Ser Tyr Arg Met Tyr Lys Thr Ala Leu Ile
-25 -20 -15 ttg gca gcc ctc ttg ggc tcg gga cag gca cag cag gtc gga
acc tcg 96Leu Ala Ala Leu Leu Gly Ser Gly Gln Ala Gln Gln Val Gly
Thr Ser -10 -5 -1 1 5 cag gcc gag gtc cat cct tcc atg acg tgg cag
tcg tgt aca gcg ggt 144Gln Ala Glu Val His Pro Ser Met Thr Trp Gln
Ser Cys Thr Ala Gly 10 15 20 ggt tcg tgt acc aca aac aac ggt aaa
gtc gtg atc gat gca aac tgg 192Gly Ser Cys Thr Thr Asn Asn Gly Lys
Val Val Ile Asp Ala Asn Trp 25 30 35 agg tgg gtg cac aag gtc ggc
gac tac acc aac tgt tac aca ggc aac 240Arg Trp Val His Lys Val Gly
Asp Tyr Thr Asn Cys Tyr Thr Gly Asn 40 45 50 aca tgg gat aca acc
atc tgt ccc gac gat gcc act tgt gca tcc aac 288Thr Trp Asp Thr Thr
Ile Cys Pro Asp Asp Ala Thr Cys Ala Ser Asn 55 60 65 70 tgt gca ctc
gag ggt gcc aac tat gag tcg acg tac gga gtg acc gcc 336Cys Ala Leu
Glu Gly Ala Asn Tyr Glu Ser Thr Tyr Gly Val Thr Ala 75 80 85 tcc
gga aac tcg ctc agg ctc aac ttc gtc aca act tcc cag cag aag 384Ser
Gly Asn Ser Leu Arg Leu Asn Phe Val Thr Thr Ser Gln Gln Lys 90 95
100 aac atc ggc tcg cgg ttg tat atg atg aaa gac gat tcc act tac gag
432Asn Ile Gly Ser Arg Leu Tyr Met Met Lys Asp Asp Ser Thr Tyr Glu
105 110 115 atg ttc aag ctc ctc aac cag gaa ttc act ttc gat gtc gac
gtc tcc 480Met Phe Lys Leu Leu Asn Gln Glu Phe Thr Phe Asp Val Asp
Val Ser 120 125 130 aac ctc cct tgt ggc ttg aac gga gcg ctc tac ttc
gtc gcc atg gat 528Asn Leu Pro Cys Gly Leu Asn Gly Ala Leu Tyr Phe
Val Ala Met Asp 135 140 145 150 gcg gat gga ggc atg tcc aag tat cct
acc aac aaa gca gga gcc aag 576Ala Asp Gly Gly Met Ser Lys Tyr Pro
Thr Asn Lys Ala Gly Ala Lys 155 160 165 tat ggt aca ggt tac tgt gat
tcc cag tgt ccc agg gat ctc aag ttc 624Tyr Gly Thr Gly Tyr Cys Asp
Ser Gln Cys Pro Arg Asp Leu Lys Phe 170 175 180 atc aac ggt cag gcc
aac gtc gag ggt tgg cag cct tcg tcg aac gat 672Ile Asn Gly Gln Ala
Asn Val Glu Gly Trp Gln Pro Ser Ser Asn Asp 185 190 195 gcc aac gca
ggt acc ggc aac cac ggt tcc tgt tgt gcc gaa atg gac 720Ala Asn Ala
Gly Thr Gly Asn His Gly Ser Cys Cys Ala Glu Met Asp 200 205 210 att
tgg gaa gcg aac tcg atc tcg acg gcg ttc act cct cac ccg tgt 768Ile
Trp Glu Ala Asn Ser Ile Ser Thr Ala Phe Thr Pro His Pro Cys 215 220
225 230 gat aca ccc gga cag gtg atg tgt aca ggc gac gcc tgt ggc gga
acc 816Asp Thr Pro Gly Gln Val Met Cys Thr Gly Asp Ala Cys Gly Gly
Thr 235 240 245 tac tcg tcg gat cga tat ggc ggt acg tgt gac ccc gac
ggc tgt gac 864Tyr Ser Ser Asp Arg Tyr Gly Gly Thr Cys Asp Pro Asp
Gly Cys Asp 250 255 260 ttc aac tcc ttc agg cag ggc aac aaa aca ttc
tat gga cct ggc atg 912Phe Asn Ser Phe Arg Gln Gly Asn Lys Thr Phe
Tyr Gly Pro Gly Met 265 270 275 acg gtg gat aca aag tcg aaa ttc aca
gtc gtc act cag ttc atc acc 960Thr Val Asp Thr Lys Ser Lys Phe Thr
Val Val Thr Gln Phe Ile Thr 280 285 290 gac gat ggt acg tcc tcg ggt
acc ttg aag gag atc aaa agg ttc tat 1008Asp Asp Gly Thr Ser Ser Gly
Thr Leu Lys Glu Ile Lys Arg Phe Tyr 295 300 305 310 gtc cag aac gga
aag gtc atc ccg aac tcg gag tcc acg tgg aca gga 1056Val Gln Asn Gly
Lys Val Ile Pro Asn Ser Glu Ser Thr Trp Thr Gly 315 320 325 gtg tcg
ggt aac tcc atc act acg gag tat tgt aca gcc cag aag tcg 1104Val Ser
Gly Asn Ser Ile Thr Thr Glu Tyr Cys Thr Ala Gln Lys Ser 330 335 340
ctc ttc cag gat cag aac gtc ttc gag aaa cat gga ggc ttg gaa gga
1152Leu Phe Gln Asp Gln Asn Val Phe Glu Lys His Gly Gly Leu Glu Gly
345 350 355 atg ggt gcc gca ttg gcc cag ggt atg gtc ctc gtc atg tcc
ttg tgg 1200Met Gly Ala Ala Leu Ala Gln Gly Met Val Leu Val Met Ser
Leu Trp 360 365 370 gac gac cac tcg gcc aac atg ctc tgg ttg gat tcc
aac tac ccc acc 1248Asp Asp His Ser Ala Asn Met Leu Trp Leu Asp Ser
Asn Tyr Pro Thr 375 380 385 390 act gcc tcg tcc acg aca ccg ggt gtc
gca cgc gga act tgt gat atc 1296Thr Ala Ser Ser Thr Thr Pro Gly Val
Ala Arg Gly Thr Cys Asp Ile 395 400 405 tcc tcg gga gtg cct gca gac
gtc gag gcg aac cat ccc gac gcc tac 1344Ser Ser Gly Val Pro Ala Asp
Val Glu Ala Asn His Pro Asp Ala Tyr 410 415 420 gtg gtc tac tcg aac
att aag gtg gga ccc atc ggt tcg aca ttc aac 1392Val Val Tyr Ser Asn
Ile Lys Val Gly Pro Ile Gly Ser Thr Phe Asn 425 430 435 tcc gga ggc
tcg aac cct gga ggc gga acg acc act act aca acg act 1440Ser Gly Gly
Ser Asn Pro Gly Gly Gly Thr Thr Thr Thr Thr Thr Thr 440 445 450 cag
ccg aca aca aca act acc aca gca ggc aac cct gga ggt aca ggt 1488Gln
Pro Thr Thr Thr Thr Thr Thr Ala Gly Asn Pro Gly Gly Thr Gly 455 460
465 470 gtg gcc cag cac tac gga cag tgt ggc ggt atc gga tgg aca gga
cct 1536Val Ala Gln His Tyr Gly Gln Cys Gly Gly Ile Gly Trp Thr Gly
Pro 475 480 485 act act tgt gca tcg cct tat acc tgt cag aaa ttg aac
gac tac tac 1584Thr Thr Cys Ala Ser Pro Tyr Thr Cys Gln Lys Leu Asn
Asp Tyr Tyr 490 495 500 tcg cag tgt ttg taa 1599Ser Gln Cys Leu 505
4532PRTAspergillus fumigatus 4Met Leu Ala Ser Thr Phe Ser Tyr Arg
Met Tyr Lys Thr Ala Leu Ile -25 -20 -15 Leu Ala Ala Leu Leu Gly Ser
Gly Gln Ala Gln Gln Val Gly Thr Ser -10 -5 -1 1 5 Gln Ala Glu Val
His Pro Ser Met Thr Trp Gln Ser Cys Thr Ala Gly 10 15 20 Gly Ser
Cys Thr Thr Asn Asn Gly Lys Val Val Ile Asp Ala Asn Trp 25 30 35
Arg Trp Val His Lys Val Gly Asp Tyr Thr Asn Cys Tyr Thr Gly Asn 40
45 50 Thr Trp Asp Thr Thr Ile Cys Pro Asp Asp Ala Thr Cys Ala Ser
Asn 55 60 65 70 Cys Ala Leu Glu Gly Ala Asn Tyr Glu Ser Thr Tyr Gly
Val Thr Ala 75 80 85 Ser Gly Asn Ser Leu Arg Leu Asn Phe Val Thr
Thr Ser Gln Gln Lys 90 95 100 Asn Ile Gly Ser Arg Leu Tyr Met Met
Lys Asp Asp Ser Thr Tyr Glu 105 110 115 Met Phe Lys Leu Leu Asn Gln
Glu Phe Thr Phe Asp Val Asp Val Ser 120 125 130 Asn Leu Pro Cys Gly
Leu Asn Gly Ala Leu Tyr Phe Val Ala Met Asp 135 140 145 150 Ala Asp
Gly Gly Met Ser Lys Tyr Pro Thr Asn Lys Ala Gly Ala Lys 155 160 165
Tyr Gly Thr Gly Tyr Cys Asp Ser Gln Cys Pro Arg Asp Leu Lys Phe 170
175 180 Ile Asn Gly Gln Ala Asn Val Glu Gly Trp Gln Pro Ser Ser Asn
Asp 185 190 195 Ala Asn Ala Gly Thr Gly Asn His Gly Ser Cys Cys Ala
Glu Met Asp 200 205 210 Ile Trp Glu Ala Asn Ser Ile Ser Thr Ala Phe
Thr Pro His Pro Cys 215 220 225 230 Asp Thr Pro Gly Gln Val Met Cys
Thr Gly Asp Ala Cys Gly Gly Thr 235 240 245 Tyr Ser Ser Asp Arg Tyr
Gly Gly Thr Cys Asp Pro Asp Gly Cys Asp 250 255 260 Phe Asn Ser Phe
Arg Gln Gly Asn Lys Thr Phe Tyr Gly Pro Gly Met 265 270 275 Thr Val
Asp Thr Lys Ser Lys Phe Thr Val Val Thr Gln Phe Ile Thr 280 285 290
Asp Asp Gly Thr Ser Ser Gly Thr Leu Lys Glu Ile Lys Arg Phe Tyr 295
300 305 310 Val Gln Asn Gly Lys Val Ile Pro Asn Ser Glu Ser Thr Trp
Thr Gly 315 320 325 Val Ser Gly Asn Ser Ile Thr Thr Glu Tyr Cys Thr
Ala Gln Lys Ser 330 335 340 Leu Phe Gln Asp Gln Asn Val Phe Glu Lys
His Gly Gly Leu Glu Gly 345 350 355 Met Gly Ala Ala Leu Ala Gln Gly
Met Val Leu Val Met Ser Leu Trp 360 365 370 Asp Asp His Ser Ala Asn
Met Leu Trp Leu Asp Ser Asn Tyr Pro Thr 375 380 385 390 Thr Ala Ser
Ser Thr Thr Pro Gly Val Ala Arg Gly Thr Cys Asp Ile 395 400 405 Ser
Ser Gly Val Pro Ala Asp Val Glu Ala Asn His Pro Asp Ala Tyr 410 415
420 Val Val Tyr Ser Asn Ile Lys Val Gly Pro Ile Gly Ser Thr Phe Asn
425 430 435 Ser Gly Gly Ser Asn Pro Gly Gly Gly Thr Thr Thr Thr Thr
Thr Thr 440 445 450 Gln Pro Thr Thr Thr Thr Thr Thr Ala Gly Asn Pro
Gly Gly Thr Gly 455 460 465 470 Val Ala Gln His Tyr Gly Gln Cys Gly
Gly Ile Gly Trp Thr Gly Pro 475 480 485 Thr Thr Cys Ala Ser Pro Tyr
Thr Cys Gln Lys Leu Asn Asp Tyr Tyr 490 495 500 Ser Gln Cys Leu 505
51676DNATrichoderma
reeseiexon(1)..(461)sig_peptide(1)..(17)CDS(1)..(461)mat_peptide(52)..(16-
73)Intron(462)..(529)exon(530)..(1226)CDS(530)..(1226)Intron(1227)..(1289)-
exon(1290)..(1673)CDS(1290)..(1673) 5atg tat cgg aag ttg gcc gtc
atc tcg gcc ttc ttg gcc aca gct cgt 48Met Tyr Arg Lys Leu Ala Val
Ile Ser Ala Phe Leu Ala Thr Ala Arg -15 -10 -5 gct cag tcg gcc tgc
act ctc caa tcg gag act cac ccg cct ctg aca 96Ala Gln Ser Ala Cys
Thr Leu Gln Ser Glu Thr His Pro Pro Leu Thr -1 1 5 10 15 tgg cag
aaa tgc tcg tct ggt ggc acg tgc act caa cag aca ggc tcc 144Trp Gln
Lys Cys Ser Ser Gly Gly Thr Cys Thr Gln Gln Thr Gly Ser 20 25 30
gtg gtc atc gac gcc aac tgg cgc tgg act cac gct acg aac agc agc
192Val Val Ile Asp Ala Asn Trp Arg Trp Thr His Ala Thr Asn Ser Ser
35 40 45 acg aac tgc tac gat ggc aac act tgg agc tcg acc cta tgt
cct gac 240Thr Asn Cys Tyr Asp Gly Asn Thr Trp Ser Ser Thr Leu Cys
Pro Asp 50 55 60 aac gag acc tgc gcg aag aac tgc tgt ctg gac ggt
gcc gcc tac gcg 288Asn Glu Thr Cys Ala Lys Asn Cys Cys Leu Asp Gly
Ala Ala Tyr Ala 65 70 75 tcc acg tac gga gtt acc acg agc ggt aac
agc ctc tcc att ggc ttt 336Ser Thr Tyr Gly Val Thr Thr Ser Gly Asn
Ser Leu Ser Ile Gly Phe 80 85 90 95 gtc acc cag tct gcg cag aag aac
gtt ggc gct cgc ctt tac ctt atg 384Val Thr Gln Ser Ala Gln Lys Asn
Val Gly Ala Arg Leu Tyr Leu Met 100 105 110 gcg agc gac acg acc tac
cag gaa ttc acc ctg ctt ggc aac gag ttc 432Ala Ser Asp Thr Thr Tyr
Gln Glu Phe Thr Leu Leu Gly Asn Glu Phe 115 120 125 tct ttc gat gtt
gat gtt tcg cag ctg cc gtaagtgact taccatgaac 481Ser Phe Asp Val Asp
Val Ser Gln Leu Pro 130 135 ccctgacgct atcttcttgt tggctcccag
ctgactggcc aattcaag g tgc ggc 536
Cys Gly ttg aac gga gct ctc tac ttc gtg tcc atg gac gcg gat ggt ggc
gtg 584Leu Asn Gly Ala Leu Tyr Phe Val Ser Met Asp Ala Asp Gly Gly
Val 140 145 150 155 agc aag tat ccc acc aac acc gct ggc gcc aag tac
ggc acg ggg tac 632Ser Lys Tyr Pro Thr Asn Thr Ala Gly Ala Lys Tyr
Gly Thr Gly Tyr 160 165 170 tgt gac agc cag tgt ccc cgc gat ctg aag
ttc atc aat ggc cag gcc 680Cys Asp Ser Gln Cys Pro Arg Asp Leu Lys
Phe Ile Asn Gly Gln Ala 175 180 185 aac gtt gag ggc tgg gag ccg tca
tcc aac aac gcg aac acg ggc att 728Asn Val Glu Gly Trp Glu Pro Ser
Ser Asn Asn Ala Asn Thr Gly Ile 190 195 200 gga gga cac gga agc tgc
tgc tct gag atg gat atc tgg gag gcc aac 776Gly Gly His Gly Ser Cys
Cys Ser Glu Met Asp Ile Trp Glu Ala Asn 205 210 215 tcc atc tcc gag
gct ctt acc ccc cac cct tgc acg act gtc ggc cag 824Ser Ile Ser Glu
Ala Leu Thr Pro His Pro Cys Thr Thr Val Gly Gln 220 225 230 235 gag
atc tgc gag ggt gat ggg tgc ggc gga act tac tcc gat aac aga 872Glu
Ile Cys Glu Gly Asp Gly Cys Gly Gly Thr Tyr Ser Asp Asn Arg 240 245
250 tat ggc ggc act tgc gat ccc gat ggc tgc gac tgg aac cca tac cgc
920Tyr Gly Gly Thr Cys Asp Pro Asp Gly Cys Asp Trp Asn Pro Tyr Arg
255 260 265 ctg ggc aac acc agc ttc tac ggc cct ggc tca agc ttt acc
ctc gat 968Leu Gly Asn Thr Ser Phe Tyr Gly Pro Gly Ser Ser Phe Thr
Leu Asp 270 275 280 acc acc aag aaa ttg acc gtt gtc acc cag ttc gag
acg tcg ggt gcc 1016Thr Thr Lys Lys Leu Thr Val Val Thr Gln Phe Glu
Thr Ser Gly Ala 285 290 295 atc aac cga tac tat gtc cag aat ggc gtc
act ttc cag cag ccc aac 1064Ile Asn Arg Tyr Tyr Val Gln Asn Gly Val
Thr Phe Gln Gln Pro Asn 300 305 310 315 gcc gag ctt ggt agt tac tct
ggc aac gag ctc aac gat gat tac tgc 1112Ala Glu Leu Gly Ser Tyr Ser
Gly Asn Glu Leu Asn Asp Asp Tyr Cys 320 325 330 aca gct gag gag gca
gaa ttc ggc gga tcc tct ttc tca gac aag ggc 1160Thr Ala Glu Glu Ala
Glu Phe Gly Gly Ser Ser Phe Ser Asp Lys Gly 335 340 345 ggc ctg act
cag ttc aag aag gct acc tct ggc ggc atg gtt ctg gtc 1208Gly Leu Thr
Gln Phe Lys Lys Ala Thr Ser Gly Gly Met Val Leu Val 350 355 360 atg
agt ctg tgg gat gat gtgagtttga tggacaaaca tgcgcgttga 1256Met Ser
Leu Trp Asp Asp 365 caaagagtca agcagctgac tgagatgtta cag tac tac
gcc aac atg ctg tgg 1310 Tyr Tyr Ala Asn Met Leu Trp 370 375 ctg
gac tcc acc tac ccg aca aac gag acc tcc tcc aca ccc ggt gcc 1358Leu
Asp Ser Thr Tyr Pro Thr Asn Glu Thr Ser Ser Thr Pro Gly Ala 380 385
390 gtg cgc gga agc tgc tcc acc agc tcc ggt gtc cct gct cag gtc gaa
1406Val Arg Gly Ser Cys Ser Thr Ser Ser Gly Val Pro Ala Gln Val Glu
395 400 405 tct cag tct ccc aac gcc aag gtc acc ttc tcc aac atc aag
ttc gga 1454Ser Gln Ser Pro Asn Ala Lys Val Thr Phe Ser Asn Ile Lys
Phe Gly 410 415 420 ccc att ggc agc acc ggc aac cct agc ggc ggc aac
cct ccc ggc gga 1502Pro Ile Gly Ser Thr Gly Asn Pro Ser Gly Gly Asn
Pro Pro Gly Gly 425 430 435 440 aac ccg cct ggc acc acc acc acc cgc
cgc cca gcc act acc act gga 1550Asn Pro Pro Gly Thr Thr Thr Thr Arg
Arg Pro Ala Thr Thr Thr Gly 445 450 455 agc tct ccc gga cct acc cag
tct cac tac ggc cag tgc ggc ggt att 1598Ser Ser Pro Gly Pro Thr Gln
Ser His Tyr Gly Gln Cys Gly Gly Ile 460 465 470 ggc tac agc ggc ccc
acg gtc tgc gcc agc ggc aca act tgc cag gtc 1646Gly Tyr Ser Gly Pro
Thr Val Cys Ala Ser Gly Thr Thr Cys Gln Val 475 480 485 ctg aac cct
tac tac tct cag tgc ctg taa 1676Leu Asn Pro Tyr Tyr Ser Gln Cys Leu
490 495 6514PRTTrichoderma reesei 6Met Tyr Arg Lys Leu Ala Val Ile
Ser Ala Phe Leu Ala Thr Ala Arg -15 -10 -5 Ala Gln Ser Ala Cys Thr
Leu Gln Ser Glu Thr His Pro Pro Leu Thr -1 1 5 10 15 Trp Gln Lys
Cys Ser Ser Gly Gly Thr Cys Thr Gln Gln Thr Gly Ser 20 25 30 Val
Val Ile Asp Ala Asn Trp Arg Trp Thr His Ala Thr Asn Ser Ser 35 40
45 Thr Asn Cys Tyr Asp Gly Asn Thr Trp Ser Ser Thr Leu Cys Pro Asp
50 55 60 Asn Glu Thr Cys Ala Lys Asn Cys Cys Leu Asp Gly Ala Ala
Tyr Ala 65 70 75 Ser Thr Tyr Gly Val Thr Thr Ser Gly Asn Ser Leu
Ser Ile Gly Phe 80 85 90 95 Val Thr Gln Ser Ala Gln Lys Asn Val Gly
Ala Arg Leu Tyr Leu Met 100 105 110 Ala Ser Asp Thr Thr Tyr Gln Glu
Phe Thr Leu Leu Gly Asn Glu Phe 115 120 125 Ser Phe Asp Val Asp Val
Ser Gln Leu Pro Cys Gly Leu Asn Gly Ala 130 135 140 Leu Tyr Phe Val
Ser Met Asp Ala Asp Gly Gly Val Ser Lys Tyr Pro 145 150 155 Thr Asn
Thr Ala Gly Ala Lys Tyr Gly Thr Gly Tyr Cys Asp Ser Gln 160 165 170
175 Cys Pro Arg Asp Leu Lys Phe Ile Asn Gly Gln Ala Asn Val Glu Gly
180 185 190 Trp Glu Pro Ser Ser Asn Asn Ala Asn Thr Gly Ile Gly Gly
His Gly 195 200 205 Ser Cys Cys Ser Glu Met Asp Ile Trp Glu Ala Asn
Ser Ile Ser Glu 210 215 220 Ala Leu Thr Pro His Pro Cys Thr Thr Val
Gly Gln Glu Ile Cys Glu 225 230 235 Gly Asp Gly Cys Gly Gly Thr Tyr
Ser Asp Asn Arg Tyr Gly Gly Thr 240 245 250 255 Cys Asp Pro Asp Gly
Cys Asp Trp Asn Pro Tyr Arg Leu Gly Asn Thr 260 265 270 Ser Phe Tyr
Gly Pro Gly Ser Ser Phe Thr Leu Asp Thr Thr Lys Lys 275 280 285 Leu
Thr Val Val Thr Gln Phe Glu Thr Ser Gly Ala Ile Asn Arg Tyr 290 295
300 Tyr Val Gln Asn Gly Val Thr Phe Gln Gln Pro Asn Ala Glu Leu Gly
305 310 315 Ser Tyr Ser Gly Asn Glu Leu Asn Asp Asp Tyr Cys Thr Ala
Glu Glu 320 325 330 335 Ala Glu Phe Gly Gly Ser Ser Phe Ser Asp Lys
Gly Gly Leu Thr Gln 340 345 350 Phe Lys Lys Ala Thr Ser Gly Gly Met
Val Leu Val Met Ser Leu Trp 355 360 365 Asp Asp Tyr Tyr Ala Asn Met
Leu Trp Leu Asp Ser Thr Tyr Pro Thr 370 375 380 Asn Glu Thr Ser Ser
Thr Pro Gly Ala Val Arg Gly Ser Cys Ser Thr 385 390 395 Ser Ser Gly
Val Pro Ala Gln Val Glu Ser Gln Ser Pro Asn Ala Lys 400 405 410 415
Val Thr Phe Ser Asn Ile Lys Phe Gly Pro Ile Gly Ser Thr Gly Asn 420
425 430 Pro Ser Gly Gly Asn Pro Pro Gly Gly Asn Pro Pro Gly Thr Thr
Thr 435 440 445 Thr Arg Arg Pro Ala Thr Thr Thr Gly Ser Ser Pro Gly
Pro Thr Gln 450 455 460 Ser His Tyr Gly Gln Cys Gly Gly Ile Gly Tyr
Ser Gly Pro Thr Val 465 470 475 Cys Ala Ser Gly Thr Thr Cys Gln Val
Leu Asn Pro Tyr Tyr Ser Gln 480 485 490 495 Cys Leu
71374DNAThermoascus
aurantiacussig_peptide(1)..(17)CDS(1)..(1371)mat_peptide(52)..(1371)
7atg tat cag cgc gct ctt ctc ttc tct ttc ttc ctc tcc gcc gcc cgc
48Met Tyr Gln Arg Ala Leu Leu Phe Ser Phe Phe Leu Ser Ala Ala Arg
-15 -10 -5 gcg cag cag gcc ggt acc cta acc gca gag aat cac cct tcc
ctg acc 96Ala Gln Gln Ala Gly Thr Leu Thr Ala Glu Asn His Pro Ser
Leu Thr -1 1 5 10 15 tgg cag caa tgc tcc agc ggc ggt agt tgt acc
acg cag aat gga aaa 144Trp Gln Gln Cys Ser Ser Gly Gly Ser Cys Thr
Thr Gln Asn Gly Lys 20 25 30 gtc gtt atc gat gcg aac tgg cgt tgg
gtc cat acc acc tct gga tac 192Val Val Ile Asp Ala Asn Trp Arg Trp
Val His Thr Thr Ser Gly Tyr 35 40 45 acc aac tgc tac acg ggc aat
acg tgg gac acc agt atc tgt ccc gac 240Thr Asn Cys Tyr Thr Gly Asn
Thr Trp Asp Thr Ser Ile Cys Pro Asp 50 55 60 gac gtg acc tgc gct
cag aat tgt gcc ttg gat gga gcg gat tac agt 288Asp Val Thr Cys Ala
Gln Asn Cys Ala Leu Asp Gly Ala Asp Tyr Ser 65 70 75 ggc acc tat
ggt gtt acg acc agt ggc aac gcc ctg aga ctg aac ttt 336Gly Thr Tyr
Gly Val Thr Thr Ser Gly Asn Ala Leu Arg Leu Asn Phe 80 85 90 95 gtc
acc caa agc tca ggg aag aac att ggc tcg cgc ctg tac ctg ctg 384Val
Thr Gln Ser Ser Gly Lys Asn Ile Gly Ser Arg Leu Tyr Leu Leu 100 105
110 cag gac gac acc act tat cag atc ttc aag ctg ctg ggt cag gag ttt
432Gln Asp Asp Thr Thr Tyr Gln Ile Phe Lys Leu Leu Gly Gln Glu Phe
115 120 125 acc ttc gat gtc gac gtc tcc aat ctc cct tgc ggg ctg aac
ggc gcc 480Thr Phe Asp Val Asp Val Ser Asn Leu Pro Cys Gly Leu Asn
Gly Ala 130 135 140 ctc tac ttt gtg gcc atg gac gcc gac ggc gga ttg
tcc aaa tac cct 528Leu Tyr Phe Val Ala Met Asp Ala Asp Gly Gly Leu
Ser Lys Tyr Pro 145 150 155 ggc aac aag gca ggc gct aag tat ggc act
ggt tac tgc gac tct cag 576Gly Asn Lys Ala Gly Ala Lys Tyr Gly Thr
Gly Tyr Cys Asp Ser Gln 160 165 170 175 tgc cct cgg gat ctc aag ttc
atc aac ggt cag gcc aac gtt gaa ggc 624Cys Pro Arg Asp Leu Lys Phe
Ile Asn Gly Gln Ala Asn Val Glu Gly 180 185 190 tgg cag ccg tct gcc
aac gac cca aat gcc ggc gtt ggt aac cac ggt 672Trp Gln Pro Ser Ala
Asn Asp Pro Asn Ala Gly Val Gly Asn His Gly 195 200 205 tcc tgc tgc
gct gag atg gat gtc tgg gaa gcc aac agc atc tct act 720Ser Cys Cys
Ala Glu Met Asp Val Trp Glu Ala Asn Ser Ile Ser Thr 210 215 220 gcg
gtg acg cct cac cca tgc gac acc ccc ggc cag acc atg tgc cag 768Ala
Val Thr Pro His Pro Cys Asp Thr Pro Gly Gln Thr Met Cys Gln 225 230
235 gga gac gac tgt ggt gga acc tac tcc tcc act cga tat gct ggt acc
816Gly Asp Asp Cys Gly Gly Thr Tyr Ser Ser Thr Arg Tyr Ala Gly Thr
240 245 250 255 tgc gac cct gat ggc tgc gac ttc aat cct tac cgc cag
ggc aac cac 864Cys Asp Pro Asp Gly Cys Asp Phe Asn Pro Tyr Arg Gln
Gly Asn His 260 265 270 tcg ttc tac ggc ccc ggg aag atc gtc gac act
agc tcc aaa ttc acc 912Ser Phe Tyr Gly Pro Gly Lys Ile Val Asp Thr
Ser Ser Lys Phe Thr 275 280 285 gtc gtc acc cag ttc atc acc gac gac
ggg acc ccc tcc ggc acc ctg 960Val Val Thr Gln Phe Ile Thr Asp Asp
Gly Thr Pro Ser Gly Thr Leu 290 295 300 acg gag atc aaa cgc ttc tac
gtc cag aac ggc aag gtg atc ccc cag 1008Thr Glu Ile Lys Arg Phe Tyr
Val Gln Asn Gly Lys Val Ile Pro Gln 305 310 315 tcg gag tcg acg atc
agc ggc gtc acc ggc aac tca atc acc acc gag 1056Ser Glu Ser Thr Ile
Ser Gly Val Thr Gly Asn Ser Ile Thr Thr Glu 320 325 330 335 tat tgc
acg gcc cag aag gcc gcc ttc ggc gac aac acc ggc ttc ttc 1104Tyr Cys
Thr Ala Gln Lys Ala Ala Phe Gly Asp Asn Thr Gly Phe Phe 340 345 350
acg cac ggc ggg ctt cag aag atc agt cag gct ctg gct cag ggc atg
1152Thr His Gly Gly Leu Gln Lys Ile Ser Gln Ala Leu Ala Gln Gly Met
355 360 365 gtc ctc gtc atg agc ctg tgg gac gat cac gcc gcc aac atg
ctc tgg 1200Val Leu Val Met Ser Leu Trp Asp Asp His Ala Ala Asn Met
Leu Trp 370 375 380 ctg gac agc acc tac ccg act gat gcg gac ccg gac
acc cct ggc gtc 1248Leu Asp Ser Thr Tyr Pro Thr Asp Ala Asp Pro Asp
Thr Pro Gly Val 385 390 395 gcg cgc ggt acc tgc ccc acg acc tcc ggc
gtc ccg gcc gac gtt gag 1296Ala Arg Gly Thr Cys Pro Thr Thr Ser Gly
Val Pro Ala Asp Val Glu 400 405 410 415 tcg cag aac ccc aat tca tat
gtt atc tac tcc aac atc aag gtc gga 1344Ser Gln Asn Pro Asn Ser Tyr
Val Ile Tyr Ser Asn Ile Lys Val Gly 420 425 430 ccc atc aac tcg acc
ttc acc gcc aac taa 1374Pro Ile Asn Ser Thr Phe Thr Ala Asn 435 440
8457PRTThermoascus aurantiacus 8Met Tyr Gln Arg Ala Leu Leu Phe Ser
Phe Phe Leu Ser Ala Ala Arg -15 -10 -5 Ala Gln Gln Ala Gly Thr Leu
Thr Ala Glu Asn His Pro Ser Leu Thr -1 1 5 10 15 Trp Gln Gln Cys
Ser Ser Gly Gly Ser Cys Thr Thr Gln Asn Gly Lys 20 25 30 Val Val
Ile Asp Ala Asn Trp Arg Trp Val His Thr Thr Ser Gly Tyr 35 40 45
Thr Asn Cys Tyr Thr Gly Asn Thr Trp Asp Thr Ser Ile Cys Pro Asp 50
55 60 Asp Val Thr Cys Ala Gln Asn Cys Ala Leu Asp Gly Ala Asp Tyr
Ser 65 70 75 Gly Thr Tyr Gly Val Thr Thr Ser Gly Asn Ala Leu Arg
Leu Asn Phe 80 85 90 95 Val Thr Gln Ser Ser Gly Lys Asn Ile Gly Ser
Arg Leu Tyr Leu Leu 100 105 110 Gln Asp Asp Thr Thr Tyr Gln Ile Phe
Lys Leu Leu Gly Gln Glu Phe 115 120 125 Thr Phe Asp Val Asp Val Ser
Asn Leu Pro Cys Gly Leu Asn Gly Ala 130 135 140 Leu Tyr Phe Val Ala
Met Asp Ala Asp Gly Gly Leu Ser Lys Tyr Pro 145 150 155 Gly Asn Lys
Ala Gly Ala Lys Tyr Gly Thr Gly Tyr Cys Asp Ser Gln 160 165 170 175
Cys Pro Arg Asp Leu Lys Phe Ile Asn Gly Gln Ala Asn Val Glu Gly 180
185 190 Trp Gln Pro Ser Ala Asn Asp Pro Asn Ala Gly Val Gly Asn His
Gly 195 200 205 Ser Cys Cys Ala Glu Met Asp Val Trp Glu Ala Asn Ser
Ile Ser Thr 210 215 220 Ala Val Thr Pro His Pro Cys Asp Thr Pro Gly
Gln Thr Met Cys Gln 225 230 235 Gly Asp Asp Cys Gly Gly Thr Tyr Ser
Ser Thr Arg Tyr Ala Gly Thr 240 245 250 255 Cys Asp Pro Asp Gly Cys
Asp Phe Asn Pro Tyr Arg Gln Gly Asn His 260 265 270 Ser Phe Tyr Gly
Pro Gly Lys Ile Val Asp Thr Ser Ser Lys Phe Thr 275 280 285 Val Val
Thr Gln Phe Ile Thr Asp Asp Gly Thr Pro Ser Gly Thr Leu 290 295
300 Thr Glu Ile Lys Arg Phe Tyr Val Gln Asn Gly Lys Val Ile Pro Gln
305 310 315 Ser Glu Ser Thr Ile Ser Gly Val Thr Gly Asn Ser Ile Thr
Thr Glu 320 325 330 335 Tyr Cys Thr Ala Gln Lys Ala Ala Phe Gly Asp
Asn Thr Gly Phe Phe 340 345 350 Thr His Gly Gly Leu Gln Lys Ile Ser
Gln Ala Leu Ala Gln Gly Met 355 360 365 Val Leu Val Met Ser Leu Trp
Asp Asp His Ala Ala Asn Met Leu Trp 370 375 380 Leu Asp Ser Thr Tyr
Pro Thr Asp Ala Asp Pro Asp Thr Pro Gly Val 385 390 395 Ala Arg Gly
Thr Cys Pro Thr Thr Ser Gly Val Pro Ala Asp Val Glu 400 405 410 415
Ser Gln Asn Pro Asn Ser Tyr Val Ile Tyr Ser Asn Ile Lys Val Gly 420
425 430 Pro Ile Asn Ser Thr Phe Thr Ala Asn 435 440
91428DNARasamsonia
emersoniiexon(1)..(603)sig_peptide(1)..(18)CDS(1)..(603)mat_peptide(55)..-
(1425)Intron(604)..(663)exon(664)..(1425)CDS(664)..(1425) 9atg ctt
cga cgg gct ctt ctt cta tcc tct tcc gcc atc ctt gct gtc 48Met Leu
Arg Arg Ala Leu Leu Leu Ser Ser Ser Ala Ile Leu Ala Val -15 -10 -5
aag gca cag cag gcc ggc acg gcg acg gca gag aac cac ccg ccc ctg
96Lys Ala Gln Gln Ala Gly Thr Ala Thr Ala Glu Asn His Pro Pro Leu
-1 1 5 10 aca tgg cag gaa tgc acc gcc cct ggg agc tgc acc acc cag
aac ggg 144Thr Trp Gln Glu Cys Thr Ala Pro Gly Ser Cys Thr Thr Gln
Asn Gly 15 20 25 30 gcg gtc gtt ctt gat gcg aac tgg cgt tgg gtg cac
gat gtg aac gga 192Ala Val Val Leu Asp Ala Asn Trp Arg Trp Val His
Asp Val Asn Gly 35 40 45 tac acc aac tgc tac acg ggc aat acc tgg
aac ccc acg tac tgc cct 240Tyr Thr Asn Cys Tyr Thr Gly Asn Thr Trp
Asn Pro Thr Tyr Cys Pro 50 55 60 gac gac gaa acc tgc gcc cag aac
tgt gcg ctg gac ggc gcg gat tac 288Asp Asp Glu Thr Cys Ala Gln Asn
Cys Ala Leu Asp Gly Ala Asp Tyr 65 70 75 gag ggc acc tac ggc gtg
act tcg tcg ggc agc tcc ttg aag ctc aat 336Glu Gly Thr Tyr Gly Val
Thr Ser Ser Gly Ser Ser Leu Lys Leu Asn 80 85 90 ttc gtc acc ggg
tcg aac gtc gga tcc cgt ctc tac ctg ctg cag gac 384Phe Val Thr Gly
Ser Asn Val Gly Ser Arg Leu Tyr Leu Leu Gln Asp 95 100 105 110 gac
tcg acc tat cag atc ttc aag ctt ctg aac cgc gag ttt acc ttt 432Asp
Ser Thr Tyr Gln Ile Phe Lys Leu Leu Asn Arg Glu Phe Thr Phe 115 120
125 gac gtc gat gtc tcc aat ctt ccg tgc gga ttg aac ggc gct ctg tac
480Asp Val Asp Val Ser Asn Leu Pro Cys Gly Leu Asn Gly Ala Leu Tyr
130 135 140 ttt gtc gcc atg gac gcc gac ggc ggc gtg tcc aag tac ccg
aac aac 528Phe Val Ala Met Asp Ala Asp Gly Gly Val Ser Lys Tyr Pro
Asn Asn 145 150 155 aag gct ggt gcc aag tac gga acc ggg tat tgc gac
tcc caa tgc cca 576Lys Ala Gly Ala Lys Tyr Gly Thr Gly Tyr Cys Asp
Ser Gln Cys Pro 160 165 170 cgg gac ctc aag ttc atc gac ggc gag
gtatgtccag tggtaaaatc 623Arg Asp Leu Lys Phe Ile Asp Gly Glu 175
180 gatcgtctcg tgaacttctg ctgacaggtt cgatctacag gcc aac gtc gag ggc
678 Ala Asn Val Glu Gly 185 tgg cag ccg tct tcg aac aac gcc aac acc
gga att ggc gac cat ggc 726Trp Gln Pro Ser Ser Asn Asn Ala Asn Thr
Gly Ile Gly Asp His Gly 190 195 200 tcc tgc tgt gcg gag atg gat gtc
tgg gaa gcc aac agc atc tcc aat 774Ser Cys Cys Ala Glu Met Asp Val
Trp Glu Ala Asn Ser Ile Ser Asn 205 210 215 220 gcg gtc act ccg cac
ccg tgc gac acg cca ggc cag acg atg tgc tct 822Ala Val Thr Pro His
Pro Cys Asp Thr Pro Gly Gln Thr Met Cys Ser 225 230 235 ggc gat gac
tgc ggt ggc aca tac tct aac gat cgc tac gcg gga acc 870Gly Asp Asp
Cys Gly Gly Thr Tyr Ser Asn Asp Arg Tyr Ala Gly Thr 240 245 250 tgc
gat cct gac ggc tgt gac ttc aac cct tac cgc atg ggc aac act 918Cys
Asp Pro Asp Gly Cys Asp Phe Asn Pro Tyr Arg Met Gly Asn Thr 255 260
265 tct ttc tac ggg cct ggc aag atc atc gat acc acc aag cct ttc act
966Ser Phe Tyr Gly Pro Gly Lys Ile Ile Asp Thr Thr Lys Pro Phe Thr
270 275 280 gtc gtg acg cag ttc ctc act gat gat ggt acg gat act gga
act ctc 1014Val Val Thr Gln Phe Leu Thr Asp Asp Gly Thr Asp Thr Gly
Thr Leu 285 290 295 300 agc gag atc aag cgc ttc tac gtc cag aac ggc
aac gtc att ccg cag 1062Ser Glu Ile Lys Arg Phe Tyr Val Gln Asn Gly
Asn Val Ile Pro Gln 305 310 315 ccc aac tcg gac atc agt ggc gtg acc
ggc aac tcg atc acg acg gag 1110Pro Asn Ser Asp Ile Ser Gly Val Thr
Gly Asn Ser Ile Thr Thr Glu 320 325 330 ttc tgt act gct cag aag cag
gcc ttt ggc gac acg gac gac ttc tct 1158Phe Cys Thr Ala Gln Lys Gln
Ala Phe Gly Asp Thr Asp Asp Phe Ser 335 340 345 cag cac ggt ggc ctg
gcc aag atg gga gcg gcc atg cag cag ggt atg 1206Gln His Gly Gly Leu
Ala Lys Met Gly Ala Ala Met Gln Gln Gly Met 350 355 360 gtc ctg gtg
atg agt ttg tgg gac gac tac gcc gcg cag atg ctg tgg 1254Val Leu Val
Met Ser Leu Trp Asp Asp Tyr Ala Ala Gln Met Leu Trp 365 370 375 380
ctg gat tcc gac tac ccg acg gat gcg gac ccc acg acc cct ggt att
1302Leu Asp Ser Asp Tyr Pro Thr Asp Ala Asp Pro Thr Thr Pro Gly Ile
385 390 395 gcc cgt gga acg tgt ccg acg gac tcg ggc gtc cca tcg gat
gtc gag 1350Ala Arg Gly Thr Cys Pro Thr Asp Ser Gly Val Pro Ser Asp
Val Glu 400 405 410 tcg cag agc ccc aac tcc tac gtg acc tac tcg aac
atc aag ttt ggt 1398Ser Gln Ser Pro Asn Ser Tyr Val Thr Tyr Ser Asn
Ile Lys Phe Gly 415 420 425 ccg atc aac tcg acc ttc acc gct tcg tga
1428Pro Ile Asn Ser Thr Phe Thr Ala Ser 430 435 10455PRTRasamsonia
emersonii 10Met Leu Arg Arg Ala Leu Leu Leu Ser Ser Ser Ala Ile Leu
Ala Val -15 -10 -5 Lys Ala Gln Gln Ala Gly Thr Ala Thr Ala Glu Asn
His Pro Pro Leu -1 1 5 10 Thr Trp Gln Glu Cys Thr Ala Pro Gly Ser
Cys Thr Thr Gln Asn Gly 15 20 25 30 Ala Val Val Leu Asp Ala Asn Trp
Arg Trp Val His Asp Val Asn Gly 35 40 45 Tyr Thr Asn Cys Tyr Thr
Gly Asn Thr Trp Asn Pro Thr Tyr Cys Pro 50 55 60 Asp Asp Glu Thr
Cys Ala Gln Asn Cys Ala Leu Asp Gly Ala Asp Tyr 65 70 75 Glu Gly
Thr Tyr Gly Val Thr Ser Ser Gly Ser Ser Leu Lys Leu Asn 80 85 90
Phe Val Thr Gly Ser Asn Val Gly Ser Arg Leu Tyr Leu Leu Gln Asp 95
100 105 110 Asp Ser Thr Tyr Gln Ile Phe Lys Leu Leu Asn Arg Glu Phe
Thr Phe 115 120 125 Asp Val Asp Val Ser Asn Leu Pro Cys Gly Leu Asn
Gly Ala Leu Tyr 130 135 140 Phe Val Ala Met Asp Ala Asp Gly Gly Val
Ser Lys Tyr Pro Asn Asn 145 150 155 Lys Ala Gly Ala Lys Tyr Gly Thr
Gly Tyr Cys Asp Ser Gln Cys Pro 160 165 170 Arg Asp Leu Lys Phe Ile
Asp Gly Glu Ala Asn Val Glu Gly Trp Gln 175 180 185 190 Pro Ser Ser
Asn Asn Ala Asn Thr Gly Ile Gly Asp His Gly Ser Cys 195 200 205 Cys
Ala Glu Met Asp Val Trp Glu Ala Asn Ser Ile Ser Asn Ala Val 210 215
220 Thr Pro His Pro Cys Asp Thr Pro Gly Gln Thr Met Cys Ser Gly Asp
225 230 235 Asp Cys Gly Gly Thr Tyr Ser Asn Asp Arg Tyr Ala Gly Thr
Cys Asp 240 245 250 Pro Asp Gly Cys Asp Phe Asn Pro Tyr Arg Met Gly
Asn Thr Ser Phe 255 260 265 270 Tyr Gly Pro Gly Lys Ile Ile Asp Thr
Thr Lys Pro Phe Thr Val Val 275 280 285 Thr Gln Phe Leu Thr Asp Asp
Gly Thr Asp Thr Gly Thr Leu Ser Glu 290 295 300 Ile Lys Arg Phe Tyr
Val Gln Asn Gly Asn Val Ile Pro Gln Pro Asn 305 310 315 Ser Asp Ile
Ser Gly Val Thr Gly Asn Ser Ile Thr Thr Glu Phe Cys 320 325 330 Thr
Ala Gln Lys Gln Ala Phe Gly Asp Thr Asp Asp Phe Ser Gln His 335 340
345 350 Gly Gly Leu Ala Lys Met Gly Ala Ala Met Gln Gln Gly Met Val
Leu 355 360 365 Val Met Ser Leu Trp Asp Asp Tyr Ala Ala Gln Met Leu
Trp Leu Asp 370 375 380 Ser Asp Tyr Pro Thr Asp Ala Asp Pro Thr Thr
Pro Gly Ile Ala Arg 385 390 395 Gly Thr Cys Pro Thr Asp Ser Gly Val
Pro Ser Asp Val Glu Ser Gln 400 405 410 Ser Pro Asn Ser Tyr Val Thr
Tyr Ser Asn Ile Lys Phe Gly Pro Ile 415 420 425 430 Asn Ser Thr Phe
Thr Ala Ser 435 111599DNATalaromyces
leycettanussig_peptide(1)..(25)CDS(1)..(1596)mat_peptide(76)..(1596)
11atg gcc agc ctc ttc tct ttc aag atg tac aag gcc gct ctg gtc ctc
48Met Ala Ser Leu Phe Ser Phe Lys Met Tyr Lys Ala Ala Leu Val Leu
-25 -20 -15 -10 tcc tct ctc ctt gcg gcc acc cag gcc cag cag gcc ggc
acc ctg acc 96Ser Ser Leu Leu Ala Ala Thr Gln Ala Gln Gln Ala Gly
Thr Leu Thr -5 -1 1 5 acc gaa acc cat cct tct ctg acc tgg cag caa
tgc tct gcc ggc ggc 144Thr Glu Thr His Pro Ser Leu Thr Trp Gln Gln
Cys Ser Ala Gly Gly 10 15 20 agc tgc acc act cag aac ggc aag gtc
gtc atc gac gcc aac tgg cgc 192Ser Cys Thr Thr Gln Asn Gly Lys Val
Val Ile Asp Ala Asn Trp Arg 25 30 35 tgg gtt cac agc acc agc ggc
tcg aac aac tgc tac act ggc aac act 240Trp Val His Ser Thr Ser Gly
Ser Asn Asn Cys Tyr Thr Gly Asn Thr 40 45 50 55 tgg gat gct act ctc
tgc cct gac gac gtg act tgc gct gcc aac tgc 288Trp Asp Ala Thr Leu
Cys Pro Asp Asp Val Thr Cys Ala Ala Asn Cys 60 65 70 gcc ctg gac
ggc gct gac tac tcg ggc acc tac ggt gtc acc acc agc 336Ala Leu Asp
Gly Ala Asp Tyr Ser Gly Thr Tyr Gly Val Thr Thr Ser 75 80 85 ggc
aac tct ctg cgc ctg aac ttc gtc acc cag gcg tcg cag aag aac 384Gly
Asn Ser Leu Arg Leu Asn Phe Val Thr Gln Ala Ser Gln Lys Asn 90 95
100 gtc ggc tct cgt ctc tat ctg atg gag aat gac aca acc tac cag atc
432Val Gly Ser Arg Leu Tyr Leu Met Glu Asn Asp Thr Thr Tyr Gln Ile
105 110 115 ttc aag ttg ctg aac cag gag ttc acc ttt gac gtt gat gtc
tcc aac 480Phe Lys Leu Leu Asn Gln Glu Phe Thr Phe Asp Val Asp Val
Ser Asn 120 125 130 135 ctt ccc tgc ggt ctc aac ggt gct ctc tac ctg
gtt gcc atg gat gcc 528Leu Pro Cys Gly Leu Asn Gly Ala Leu Tyr Leu
Val Ala Met Asp Ala 140 145 150 gac ggc ggc atg gcc aag tac cca acc
aac aag gct ggt gcg aag tac 576Asp Gly Gly Met Ala Lys Tyr Pro Thr
Asn Lys Ala Gly Ala Lys Tyr 155 160 165 gga acc ggt tac tgc gac tcc
cag tgc cct cgc gac ctg aag ttc atc 624Gly Thr Gly Tyr Cys Asp Ser
Gln Cys Pro Arg Asp Leu Lys Phe Ile 170 175 180 aac ggt gag gcc aat
gtt gag gga tgg cag cct tct tcc aat gac ccc 672Asn Gly Glu Ala Asn
Val Glu Gly Trp Gln Pro Ser Ser Asn Asp Pro 185 190 195 aac tct ggc
att ggc aac cac ggc tct tgc tgt gct gag atg gac atc 720Asn Ser Gly
Ile Gly Asn His Gly Ser Cys Cys Ala Glu Met Asp Ile 200 205 210 215
tgg gag gcc aac agc atc tcc aat gca gtc act cct cac cct tgc gac
768Trp Glu Ala Asn Ser Ile Ser Asn Ala Val Thr Pro His Pro Cys Asp
220 225 230 acc ccg gga cag gtc atg tgc acc ggc aac aac tgt ggt ggc
act tac 816Thr Pro Gly Gln Val Met Cys Thr Gly Asn Asn Cys Gly Gly
Thr Tyr 235 240 245 agc act act cgc tat gct ggc act tgc gat cct gat
ggc tgc gac ttc 864Ser Thr Thr Arg Tyr Ala Gly Thr Cys Asp Pro Asp
Gly Cys Asp Phe 250 255 260 aac ccc tac cgc atg ggc aac cac tcc ttc
tac ggc ccc aaa cag atc 912Asn Pro Tyr Arg Met Gly Asn His Ser Phe
Tyr Gly Pro Lys Gln Ile 265 270 275 gtc gac acc agc tcc aag ttc act
gtt gtt act cag ttc ctc acc gat 960Val Asp Thr Ser Ser Lys Phe Thr
Val Val Thr Gln Phe Leu Thr Asp 280 285 290 295 gat ggc acc tcc acc
ggc acc ctc agc gag atc agg cgc ttc tac gtt 1008Asp Gly Thr Ser Thr
Gly Thr Leu Ser Glu Ile Arg Arg Phe Tyr Val 300 305 310 cag aac ggc
cag gtc atc ccc aac tcc gtg tcc acg atc agc ggc gtc 1056Gln Asn Gly
Gln Val Ile Pro Asn Ser Val Ser Thr Ile Ser Gly Val 315 320 325 tcc
ggc aac tcc atc acc acc gag ttc tgc acg gcc cag aag cag gct 1104Ser
Gly Asn Ser Ile Thr Thr Glu Phe Cys Thr Ala Gln Lys Gln Ala 330 335
340 ttc ggc gac act gat gac ttc agc aag cac ggc ggt ctg tct ggc atg
1152Phe Gly Asp Thr Asp Asp Phe Ser Lys His Gly Gly Leu Ser Gly Met
345 350 355 tcc gcc gcc ctc tcc cag ggt atg gtt ctc gtc atg agc ttg
tgg gac 1200Ser Ala Ala Leu Ser Gln Gly Met Val Leu Val Met Ser Leu
Trp Asp 360 365 370 375 gac cac gcc gcc aac atg ctc tgg ctt gac agc
acc tac ccg acc aac 1248Asp His Ala Ala Asn Met Leu Trp Leu Asp Ser
Thr Tyr Pro Thr Asn 380 385 390 gcc acc tct tcc acc ccc ggt gcc gcc
cgt ggt act tgc gac atc tcc 1296Ala Thr Ser Ser Thr Pro Gly Ala Ala
Arg Gly Thr Cys Asp Ile Ser 395 400 405 tcc ggt gtc ccc gcc gat gtt
gag tcc aac gac ccc aac gcc tac gtc 1344Ser Gly Val Pro Ala Asp Val
Glu Ser Asn Asp Pro Asn Ala Tyr Val 410 415 420 gtc tac tcc aac atc
aag gtc ggc ccg atc ggc tct acc ttc agc agc 1392Val Tyr Ser Asn Ile
Lys Val Gly Pro Ile Gly Ser Thr Phe Ser Ser 425 430 435 tct ggc tct
ggc tct agc tcc agc tcc agc acc acc acc acc acc acc 1440Ser Gly Ser
Gly Ser Ser Ser Ser Ser Ser Thr Thr Thr Thr Thr Thr 440 445 450 455
gct tcc cca acc acg acc acc tcc agc gct tcc agc acc ggc act ggc
1488Ala Ser Pro Thr Thr Thr Thr Ser Ser Ala Ser Ser Thr Gly Thr Gly
460 465 470
gtt gct cag cac tgg ggt cag tgc ggt ggc cag gga tgg acc ggt ccg
1536Val Ala Gln His Trp Gly Gln Cys Gly Gly Gln Gly Trp Thr Gly Pro
475 480 485 acc acc tgc gtt agc ccc tac acc tgc cag gag ctg aac ccc
tac tac 1584Thr Thr Cys Val Ser Pro Tyr Thr Cys Gln Glu Leu Asn Pro
Tyr Tyr 490 495 500 tac cag tgc ctg taa 1599Tyr Gln Cys Leu 505
12532PRTTalaromyces leycettanus 12Met Ala Ser Leu Phe Ser Phe Lys
Met Tyr Lys Ala Ala Leu Val Leu -25 -20 -15 -10 Ser Ser Leu Leu Ala
Ala Thr Gln Ala Gln Gln Ala Gly Thr Leu Thr -5 -1 1 5 Thr Glu Thr
His Pro Ser Leu Thr Trp Gln Gln Cys Ser Ala Gly Gly 10 15 20 Ser
Cys Thr Thr Gln Asn Gly Lys Val Val Ile Asp Ala Asn Trp Arg 25 30
35 Trp Val His Ser Thr Ser Gly Ser Asn Asn Cys Tyr Thr Gly Asn Thr
40 45 50 55 Trp Asp Ala Thr Leu Cys Pro Asp Asp Val Thr Cys Ala Ala
Asn Cys 60 65 70 Ala Leu Asp Gly Ala Asp Tyr Ser Gly Thr Tyr Gly
Val Thr Thr Ser 75 80 85 Gly Asn Ser Leu Arg Leu Asn Phe Val Thr
Gln Ala Ser Gln Lys Asn 90 95 100 Val Gly Ser Arg Leu Tyr Leu Met
Glu Asn Asp Thr Thr Tyr Gln Ile 105 110 115 Phe Lys Leu Leu Asn Gln
Glu Phe Thr Phe Asp Val Asp Val Ser Asn 120 125 130 135 Leu Pro Cys
Gly Leu Asn Gly Ala Leu Tyr Leu Val Ala Met Asp Ala 140 145 150 Asp
Gly Gly Met Ala Lys Tyr Pro Thr Asn Lys Ala Gly Ala Lys Tyr 155 160
165 Gly Thr Gly Tyr Cys Asp Ser Gln Cys Pro Arg Asp Leu Lys Phe Ile
170 175 180 Asn Gly Glu Ala Asn Val Glu Gly Trp Gln Pro Ser Ser Asn
Asp Pro 185 190 195 Asn Ser Gly Ile Gly Asn His Gly Ser Cys Cys Ala
Glu Met Asp Ile 200 205 210 215 Trp Glu Ala Asn Ser Ile Ser Asn Ala
Val Thr Pro His Pro Cys Asp 220 225 230 Thr Pro Gly Gln Val Met Cys
Thr Gly Asn Asn Cys Gly Gly Thr Tyr 235 240 245 Ser Thr Thr Arg Tyr
Ala Gly Thr Cys Asp Pro Asp Gly Cys Asp Phe 250 255 260 Asn Pro Tyr
Arg Met Gly Asn His Ser Phe Tyr Gly Pro Lys Gln Ile 265 270 275 Val
Asp Thr Ser Ser Lys Phe Thr Val Val Thr Gln Phe Leu Thr Asp 280 285
290 295 Asp Gly Thr Ser Thr Gly Thr Leu Ser Glu Ile Arg Arg Phe Tyr
Val 300 305 310 Gln Asn Gly Gln Val Ile Pro Asn Ser Val Ser Thr Ile
Ser Gly Val 315 320 325 Ser Gly Asn Ser Ile Thr Thr Glu Phe Cys Thr
Ala Gln Lys Gln Ala 330 335 340 Phe Gly Asp Thr Asp Asp Phe Ser Lys
His Gly Gly Leu Ser Gly Met 345 350 355 Ser Ala Ala Leu Ser Gln Gly
Met Val Leu Val Met Ser Leu Trp Asp 360 365 370 375 Asp His Ala Ala
Asn Met Leu Trp Leu Asp Ser Thr Tyr Pro Thr Asn 380 385 390 Ala Thr
Ser Ser Thr Pro Gly Ala Ala Arg Gly Thr Cys Asp Ile Ser 395 400 405
Ser Gly Val Pro Ala Asp Val Glu Ser Asn Asp Pro Asn Ala Tyr Val 410
415 420 Val Tyr Ser Asn Ile Lys Val Gly Pro Ile Gly Ser Thr Phe Ser
Ser 425 430 435 Ser Gly Ser Gly Ser Ser Ser Ser Ser Ser Thr Thr Thr
Thr Thr Thr 440 445 450 455 Ala Ser Pro Thr Thr Thr Thr Ser Ser Ala
Ser Ser Thr Gly Thr Gly 460 465 470 Val Ala Gln His Trp Gly Gln Cys
Gly Gly Gln Gly Trp Thr Gly Pro 475 480 485 Thr Thr Cys Val Ser Pro
Tyr Thr Cys Gln Glu Leu Asn Pro Tyr Tyr 490 495 500 Tyr Gln Cys Leu
505 131599DNATalaromyces
leycettanussig_peptide(1)..(25)CDS(1)..(1596)mat_peptide(76)..(1596)
13atg gcg tcc tct ctc tct tac agg atc tac aag aat gct ctc atc ttc
48Met Ala Ser Ser Leu Ser Tyr Arg Ile Tyr Lys Asn Ala Leu Ile Phe
-25 -20 -15 -10 tct tct ctc ctg gcc gct gcc cag ggt cag cag att ggt
acc tac cag 96Ser Ser Leu Leu Ala Ala Ala Gln Gly Gln Gln Ile Gly
Thr Tyr Gln -5 -1 1 5 acg gag acc cat ccg cct ctg acc tgg cag aca
tgc acc agc ggc ggc 144Thr Glu Thr His Pro Pro Leu Thr Trp Gln Thr
Cys Thr Ser Gly Gly 10 15 20 agt tgc acg acc aac caa ggc tcc atc
gtc ctc gat gcc aac tgg cgc 192Ser Cys Thr Thr Asn Gln Gly Ser Ile
Val Leu Asp Ala Asn Trp Arg 25 30 35 tgg gtg cac gag gtc ggc agc
acc acc aac tgc tac acc ggc aat acc 240Trp Val His Glu Val Gly Ser
Thr Thr Asn Cys Tyr Thr Gly Asn Thr 40 45 50 55 tgg gac acc tcc atc
tgc agc acg gat acg acc tgc gct cag caa tgt 288Trp Asp Thr Ser Ile
Cys Ser Thr Asp Thr Thr Cys Ala Gln Gln Cys 60 65 70 gcc gtc gat
ggt gcc gac tac gag ggc acc tat ggt atc acg acc agc 336Ala Val Asp
Gly Ala Asp Tyr Glu Gly Thr Tyr Gly Ile Thr Thr Ser 75 80 85 ggc
agc cag gtc cgc atc aac ttc gtc acc aac aac tcg aac gga aag 384Gly
Ser Gln Val Arg Ile Asn Phe Val Thr Asn Asn Ser Asn Gly Lys 90 95
100 aac gtc ggc gcg cgt gtc tac atg atg gcg gac aac acc cac tac caa
432Asn Val Gly Ala Arg Val Tyr Met Met Ala Asp Asn Thr His Tyr Gln
105 110 115 att tac cag ctg ctg aac cag gag ttc acc ttt gat gtc gac
gtg tcc 480Ile Tyr Gln Leu Leu Asn Gln Glu Phe Thr Phe Asp Val Asp
Val Ser 120 125 130 135 aac ctg cct tgc ggc ctc aac ggt gcc ctc tac
ttt gtg gtc atg gac 528Asn Leu Pro Cys Gly Leu Asn Gly Ala Leu Tyr
Phe Val Val Met Asp 140 145 150 gcc gat ggt ggt gtc tcc aag tat ccc
aac aac aag gct ggt gcc cag 576Ala Asp Gly Gly Val Ser Lys Tyr Pro
Asn Asn Lys Ala Gly Ala Gln 155 160 165 tac ggt gtc ggt tac tgc gac
tcc cag tgt ccc aga gac ctc aaa ttc 624Tyr Gly Val Gly Tyr Cys Asp
Ser Gln Cys Pro Arg Asp Leu Lys Phe 170 175 180 atc cag gga cag gcc
aac gtc gag ggc tgg caa ccg tcg tcc aac aac 672Ile Gln Gly Gln Ala
Asn Val Glu Gly Trp Gln Pro Ser Ser Asn Asn 185 190 195 gcc aat acc
ggc ctg ggc aac cac ggc tcc tgc tgt gct gaa ctg gac 720Ala Asn Thr
Gly Leu Gly Asn His Gly Ser Cys Cys Ala Glu Leu Asp 200 205 210 215
gtc tgg gag tcg aac agc atc tcc cag gcc ctc act ccc cac ccc tgc
768Val Trp Glu Ser Asn Ser Ile Ser Gln Ala Leu Thr Pro His Pro Cys
220 225 230 gac act ccc acc aat acc ctg tgc acc ggt gat agc tgc ggt
ggc aca 816Asp Thr Pro Thr Asn Thr Leu Cys Thr Gly Asp Ser Cys Gly
Gly Thr 235 240 245 tac agc agc aac cgt tat gcg ggc act tgc gat cct
gac ggc tgc gat 864Tyr Ser Ser Asn Arg Tyr Ala Gly Thr Cys Asp Pro
Asp Gly Cys Asp 250 255 260 ttc aac ccc tac cgc ttg ggc aac acc acc
ttc tac ggt cct ggc aag 912Phe Asn Pro Tyr Arg Leu Gly Asn Thr Thr
Phe Tyr Gly Pro Gly Lys 265 270 275 act att gac acc acc aaa ccc ttc
acg gtt gtg acg cag ttc atc acg 960Thr Ile Asp Thr Thr Lys Pro Phe
Thr Val Val Thr Gln Phe Ile Thr 280 285 290 295 gat gac ggc act tcc
agc ggc acc ctg tcc gaa att agg cgt ttc tat 1008Asp Asp Gly Thr Ser
Ser Gly Thr Leu Ser Glu Ile Arg Arg Phe Tyr 300 305 310 gtc cag aac
ggt gtt acg tac gcc cag ccc aac tct gac gtc agc ggt 1056Val Gln Asn
Gly Val Thr Tyr Ala Gln Pro Asn Ser Asp Val Ser Gly 315 320 325 atc
agc ggc aat gcc atc aac agt gct tac tgc act gcg gag aac acc 1104Ile
Ser Gly Asn Ala Ile Asn Ser Ala Tyr Cys Thr Ala Glu Asn Thr 330 335
340 gtc ttc aac ggt gcc ggc acc ttc gcg cag cac ggc ggc ctg gct ggc
1152Val Phe Asn Gly Ala Gly Thr Phe Ala Gln His Gly Gly Leu Ala Gly
345 350 355 atg agc cag gcc atg tcc acc ggt atg gtc ttg gtg atg agc
ctg tgg 1200Met Ser Gln Ala Met Ser Thr Gly Met Val Leu Val Met Ser
Leu Trp 360 365 370 375 gat gat tac tat gcc gac atg ctc tgg ctc gac
agc acc tac cca acc 1248Asp Asp Tyr Tyr Ala Asp Met Leu Trp Leu Asp
Ser Thr Tyr Pro Thr 380 385 390 aac gac acc gca agc acg ccc ggt gcg
gtc cgt gga acc tgc tct acg 1296Asn Asp Thr Ala Ser Thr Pro Gly Ala
Val Arg Gly Thr Cys Ser Thr 395 400 405 tcg tcc ggt gtc ccc agc cag
gtc gaa tcc gcc agc ccg aac gcc tac 1344Ser Ser Gly Val Pro Ser Gln
Val Glu Ser Ala Ser Pro Asn Ala Tyr 410 415 420 gtg acc tac tcg aac
atc aag gtt ggt ccc att ggc tcg act ttc aac 1392Val Thr Tyr Ser Asn
Ile Lys Val Gly Pro Ile Gly Ser Thr Phe Asn 425 430 435 tct ggc ggc
tct ggc tct ggc agc agc tcc agc act acc acg acc act 1440Ser Gly Gly
Ser Gly Ser Gly Ser Ser Ser Ser Thr Thr Thr Thr Thr 440 445 450 455
cac gcc agc acc acg acg acg tcc tcc gcc tcg tct acg gga act ggc
1488His Ala Ser Thr Thr Thr Thr Ser Ser Ala Ser Ser Thr Gly Thr Gly
460 465 470 gtg gcc caa cac tgg ggc cag tgt ggt gga cag ggc tgg acc
ggc cca 1536Val Ala Gln His Trp Gly Gln Cys Gly Gly Gln Gly Trp Thr
Gly Pro 475 480 485 aca acc tgc gtt tcc ccg tac act tgc cag gag ctg
aac ccg tac tac 1584Thr Thr Cys Val Ser Pro Tyr Thr Cys Gln Glu Leu
Asn Pro Tyr Tyr 490 495 500 tac cag tgt ctg tag 1599Tyr Gln Cys Leu
505 14532PRTTalaromyces leycettanus 14Met Ala Ser Ser Leu Ser Tyr
Arg Ile Tyr Lys Asn Ala Leu Ile Phe -25 -20 -15 -10 Ser Ser Leu Leu
Ala Ala Ala Gln Gly Gln Gln Ile Gly Thr Tyr Gln -5 -1 1 5 Thr Glu
Thr His Pro Pro Leu Thr Trp Gln Thr Cys Thr Ser Gly Gly 10 15 20
Ser Cys Thr Thr Asn Gln Gly Ser Ile Val Leu Asp Ala Asn Trp Arg 25
30 35 Trp Val His Glu Val Gly Ser Thr Thr Asn Cys Tyr Thr Gly Asn
Thr 40 45 50 55 Trp Asp Thr Ser Ile Cys Ser Thr Asp Thr Thr Cys Ala
Gln Gln Cys 60 65 70 Ala Val Asp Gly Ala Asp Tyr Glu Gly Thr Tyr
Gly Ile Thr Thr Ser 75 80 85 Gly Ser Gln Val Arg Ile Asn Phe Val
Thr Asn Asn Ser Asn Gly Lys 90 95 100 Asn Val Gly Ala Arg Val Tyr
Met Met Ala Asp Asn Thr His Tyr Gln 105 110 115 Ile Tyr Gln Leu Leu
Asn Gln Glu Phe Thr Phe Asp Val Asp Val Ser 120 125 130 135 Asn Leu
Pro Cys Gly Leu Asn Gly Ala Leu Tyr Phe Val Val Met Asp 140 145 150
Ala Asp Gly Gly Val Ser Lys Tyr Pro Asn Asn Lys Ala Gly Ala Gln 155
160 165 Tyr Gly Val Gly Tyr Cys Asp Ser Gln Cys Pro Arg Asp Leu Lys
Phe 170 175 180 Ile Gln Gly Gln Ala Asn Val Glu Gly Trp Gln Pro Ser
Ser Asn Asn 185 190 195 Ala Asn Thr Gly Leu Gly Asn His Gly Ser Cys
Cys Ala Glu Leu Asp 200 205 210 215 Val Trp Glu Ser Asn Ser Ile Ser
Gln Ala Leu Thr Pro His Pro Cys 220 225 230 Asp Thr Pro Thr Asn Thr
Leu Cys Thr Gly Asp Ser Cys Gly Gly Thr 235 240 245 Tyr Ser Ser Asn
Arg Tyr Ala Gly Thr Cys Asp Pro Asp Gly Cys Asp 250 255 260 Phe Asn
Pro Tyr Arg Leu Gly Asn Thr Thr Phe Tyr Gly Pro Gly Lys 265 270 275
Thr Ile Asp Thr Thr Lys Pro Phe Thr Val Val Thr Gln Phe Ile Thr 280
285 290 295 Asp Asp Gly Thr Ser Ser Gly Thr Leu Ser Glu Ile Arg Arg
Phe Tyr 300 305 310 Val Gln Asn Gly Val Thr Tyr Ala Gln Pro Asn Ser
Asp Val Ser Gly 315 320 325 Ile Ser Gly Asn Ala Ile Asn Ser Ala Tyr
Cys Thr Ala Glu Asn Thr 330 335 340 Val Phe Asn Gly Ala Gly Thr Phe
Ala Gln His Gly Gly Leu Ala Gly 345 350 355 Met Ser Gln Ala Met Ser
Thr Gly Met Val Leu Val Met Ser Leu Trp 360 365 370 375 Asp Asp Tyr
Tyr Ala Asp Met Leu Trp Leu Asp Ser Thr Tyr Pro Thr 380 385 390 Asn
Asp Thr Ala Ser Thr Pro Gly Ala Val Arg Gly Thr Cys Ser Thr 395 400
405 Ser Ser Gly Val Pro Ser Gln Val Glu Ser Ala Ser Pro Asn Ala Tyr
410 415 420 Val Thr Tyr Ser Asn Ile Lys Val Gly Pro Ile Gly Ser Thr
Phe Asn 425 430 435 Ser Gly Gly Ser Gly Ser Gly Ser Ser Ser Ser Thr
Thr Thr Thr Thr 440 445 450 455 His Ala Ser Thr Thr Thr Thr Ser Ser
Ala Ser Ser Thr Gly Thr Gly 460 465 470 Val Ala Gln His Trp Gly Gln
Cys Gly Gly Gln Gly Trp Thr Gly Pro 475 480 485 Thr Thr Cys Val Ser
Pro Tyr Thr Cys Gln Glu Leu Asn Pro Tyr Tyr 490 495 500 Tyr Gln Cys
Leu 505 151507DNATalaromyces
byssochlamydoidesexon(1)..(603)sig_peptide(1)..(18)CDS(1)..(603)mat_pepti-
de(55)..(1504)Intron(604)..(667)exon(668)..(1235)CDS(668)..(1235)Intron(12-
36)..(1310)exon(1311)..(1504)CDS(1311)..(1504) 15atg ttt cga cgg
gct ctt ttc ctg tcc tct tcc gcc ttc ctt gct gtc 48Met Phe Arg Arg
Ala Leu Phe Leu Ser Ser Ser Ala Phe Leu Ala Val -15 -10 -5 aaa gcc
cag cag atc ggc acg gtc agt ccg gag aac cat ccg ccc ctg 96Lys Ala
Gln Gln Ile Gly Thr Val Ser Pro Glu Asn His Pro Pro Leu -1 1 5 10
gca tgg gag cag tgc act gcc cct ggg agt tgc acg act gtg aat ggt
144Ala Trp Glu Gln Cys Thr Ala Pro Gly Ser Cys Thr Thr Val Asn Gly
15 20 25 30 gcg gtc gtc ctt gat gcg aac tgg cgt tgg gtc cac aat gtt
ggg gga 192Ala Val Val Leu Asp Ala Asn Trp Arg Trp Val His Asn Val
Gly Gly 35 40 45 tac acc aac tgc tac act ggc aat acc tgg gac acc
acg tac tgc cct 240Tyr Thr Asn Cys Tyr Thr Gly Asn Thr Trp Asp Thr
Thr Tyr Cys Pro 50 55 60 gac gac gtg acc tgc gca gag aat tgt gcg
ctg gat ggc gca gat tac 288Asp Asp Val Thr Cys Ala Glu Asn Cys Ala
Leu Asp Gly Ala Asp Tyr 65 70 75 gag ggc acc tac ggc gtg acc acc
tcg ggc agc
tcc ctg aag ctc gat 336Glu Gly Thr Tyr Gly Val Thr Thr Ser Gly Ser
Ser Leu Lys Leu Asp 80 85 90 ttc gtc acc ggg tct aac gtc gga tct
cgt ctc tac ctg ttg gag aat 384Phe Val Thr Gly Ser Asn Val Gly Ser
Arg Leu Tyr Leu Leu Glu Asn 95 100 105 110 gat tcg acc tat cag atc
ttc aag ctt ctg aac cag gaa ttc acc ttt 432Asp Ser Thr Tyr Gln Ile
Phe Lys Leu Leu Asn Gln Glu Phe Thr Phe 115 120 125 gac gtc gac gtt
tcc aat ctt ccg tgc gga tta aac ggc gct ctg tac 480Asp Val Asp Val
Ser Asn Leu Pro Cys Gly Leu Asn Gly Ala Leu Tyr 130 135 140 ctt gtt
acc atg gct gct gac ggc ggg gtg tct cag tac ccg aat aac 528Leu Val
Thr Met Ala Ala Asp Gly Gly Val Ser Gln Tyr Pro Asn Asn 145 150 155
aag gcc ggc gca gcg tat gga acc ggt tat tgc gat tcc cag tgt cca
576Lys Ala Gly Ala Ala Tyr Gly Thr Gly Tyr Cys Asp Ser Gln Cys Pro
160 165 170 agg gac ttg aag ttt atc gat ggc cag gtatgtagag
ctgtaatcac 623Arg Asp Leu Lys Phe Ile Asp Gly Gln 175 180
ccatgttgtg aaatcactct cctactgaca tggtcgattt atag gcc aac gtt gag
679 Ala Asn Val Glu 185 ggc tgg cag ccg tct tcg aac aac gcc aat aca
ggt att ggc aac cat 727Gly Trp Gln Pro Ser Ser Asn Asn Ala Asn Thr
Gly Ile Gly Asn His 190 195 200 ggc tcc tgc tgt gcg gag atg gat atc
tgg gaa gcc aac agc atc tcc 775Gly Ser Cys Cys Ala Glu Met Asp Ile
Trp Glu Ala Asn Ser Ile Ser 205 210 215 aat gcg gtg act ccg cac cca
tgc gac aca ccc ggc cag aca atg tgc 823Asn Ala Val Thr Pro His Pro
Cys Asp Thr Pro Gly Gln Thr Met Cys 220 225 230 235 gag ggg aac gac
tgt ggt ggc acg tat tcc acc aat cgc tat gca ggc 871Glu Gly Asn Asp
Cys Gly Gly Thr Tyr Ser Thr Asn Arg Tyr Ala Gly 240 245 250 acc tgc
gat cct gac ggc tgc gac ttc aac ccc tac cgc atg ggc aac 919Thr Cys
Asp Pro Asp Gly Cys Asp Phe Asn Pro Tyr Arg Met Gly Asn 255 260 265
cat tct ttc tac ggc cct ggg gag att gtc gat act acc cag ccc ttc
967His Ser Phe Tyr Gly Pro Gly Glu Ile Val Asp Thr Thr Gln Pro Phe
270 275 280 act gtc gtg aca cag ttc ctt acc gat gat ggc acg gat act
ggc act 1015Thr Val Val Thr Gln Phe Leu Thr Asp Asp Gly Thr Asp Thr
Gly Thr 285 290 295 ctc agc gag atc aaa cgc ttc tac gtc caa aac ggg
aaa gtc att cct 1063Leu Ser Glu Ile Lys Arg Phe Tyr Val Gln Asn Gly
Lys Val Ile Pro 300 305 310 315 cag ccg aac tcc gac att gcc ggc gtg
act ggc aac tcg atc acc agc 1111Gln Pro Asn Ser Asp Ile Ala Gly Val
Thr Gly Asn Ser Ile Thr Ser 320 325 330 gag ttt tgc gat gcc cag aag
acg gct ttc ggc gac att aac aac ttt 1159Glu Phe Cys Asp Ala Gln Lys
Thr Ala Phe Gly Asp Ile Asn Asn Phe 335 340 345 gat aca cac ggc ggt
ctg gcc agt atg gga gct gcg ctg cag cag ggt 1207Asp Thr His Gly Gly
Leu Ala Ser Met Gly Ala Ala Leu Gln Gln Gly 350 355 360 atg gtt ctg
gtg atg agt ctg tgg gac g gtaggtcctt gggagacacc 1255Met Val Leu Val
Met Ser Leu Trp Asp 365 370 cggacgttct atatcaacca gaactgccag
aactgacgaa ttaaaacact tttag at 1312 Asp tac gcg gca aac atg ctg tgg
ttg gac agc att tat cca aca aat gca 1360Tyr Ala Ala Asn Met Leu Trp
Leu Asp Ser Ile Tyr Pro Thr Asn Ala 375 380 385 tct gct agc act cct
ggt gct gct cgt gga acc tgt tcg acg agc tcc 1408Ser Ala Ser Thr Pro
Gly Ala Ala Arg Gly Thr Cys Ser Thr Ser Ser 390 395 400 405 ggt gtc
cca tcg caa gtc gag tcg cag agc ccc aac gcc tac gtg acg 1456Gly Val
Pro Ser Gln Val Glu Ser Gln Ser Pro Asn Ala Tyr Val Thr 410 415 420
tac tcc aac att aaa gtt gga cca atc aac tcg acc ttc acc act tcg
1504Tyr Ser Asn Ile Lys Val Gly Pro Ile Asn Ser Thr Phe Thr Thr Ser
425 430 435 taa 1507 16455PRTTalaromyces byssochlamydoides 16Met
Phe Arg Arg Ala Leu Phe Leu Ser Ser Ser Ala Phe Leu Ala Val -15 -10
-5 Lys Ala Gln Gln Ile Gly Thr Val Ser Pro Glu Asn His Pro Pro Leu
-1 1 5 10 Ala Trp Glu Gln Cys Thr Ala Pro Gly Ser Cys Thr Thr Val
Asn Gly 15 20 25 30 Ala Val Val Leu Asp Ala Asn Trp Arg Trp Val His
Asn Val Gly Gly 35 40 45 Tyr Thr Asn Cys Tyr Thr Gly Asn Thr Trp
Asp Thr Thr Tyr Cys Pro 50 55 60 Asp Asp Val Thr Cys Ala Glu Asn
Cys Ala Leu Asp Gly Ala Asp Tyr 65 70 75 Glu Gly Thr Tyr Gly Val
Thr Thr Ser Gly Ser Ser Leu Lys Leu Asp 80 85 90 Phe Val Thr Gly
Ser Asn Val Gly Ser Arg Leu Tyr Leu Leu Glu Asn 95 100 105 110 Asp
Ser Thr Tyr Gln Ile Phe Lys Leu Leu Asn Gln Glu Phe Thr Phe 115 120
125 Asp Val Asp Val Ser Asn Leu Pro Cys Gly Leu Asn Gly Ala Leu Tyr
130 135 140 Leu Val Thr Met Ala Ala Asp Gly Gly Val Ser Gln Tyr Pro
Asn Asn 145 150 155 Lys Ala Gly Ala Ala Tyr Gly Thr Gly Tyr Cys Asp
Ser Gln Cys Pro 160 165 170 Arg Asp Leu Lys Phe Ile Asp Gly Gln Ala
Asn Val Glu Gly Trp Gln 175 180 185 190 Pro Ser Ser Asn Asn Ala Asn
Thr Gly Ile Gly Asn His Gly Ser Cys 195 200 205 Cys Ala Glu Met Asp
Ile Trp Glu Ala Asn Ser Ile Ser Asn Ala Val 210 215 220 Thr Pro His
Pro Cys Asp Thr Pro Gly Gln Thr Met Cys Glu Gly Asn 225 230 235 Asp
Cys Gly Gly Thr Tyr Ser Thr Asn Arg Tyr Ala Gly Thr Cys Asp 240 245
250 Pro Asp Gly Cys Asp Phe Asn Pro Tyr Arg Met Gly Asn His Ser Phe
255 260 265 270 Tyr Gly Pro Gly Glu Ile Val Asp Thr Thr Gln Pro Phe
Thr Val Val 275 280 285 Thr Gln Phe Leu Thr Asp Asp Gly Thr Asp Thr
Gly Thr Leu Ser Glu 290 295 300 Ile Lys Arg Phe Tyr Val Gln Asn Gly
Lys Val Ile Pro Gln Pro Asn 305 310 315 Ser Asp Ile Ala Gly Val Thr
Gly Asn Ser Ile Thr Ser Glu Phe Cys 320 325 330 Asp Ala Gln Lys Thr
Ala Phe Gly Asp Ile Asn Asn Phe Asp Thr His 335 340 345 350 Gly Gly
Leu Ala Ser Met Gly Ala Ala Leu Gln Gln Gly Met Val Leu 355 360 365
Val Met Ser Leu Trp Asp Asp Tyr Ala Ala Asn Met Leu Trp Leu Asp 370
375 380 Ser Ile Tyr Pro Thr Asn Ala Ser Ala Ser Thr Pro Gly Ala Ala
Arg 385 390 395 Gly Thr Cys Ser Thr Ser Ser Gly Val Pro Ser Gln Val
Glu Ser Gln 400 405 410 Ser Pro Asn Ala Tyr Val Thr Tyr Ser Asn Ile
Lys Val Gly Pro Ile 415 420 425 430 Asn Ser Thr Phe Thr Thr Ser 435
171353DNAMyceliophthora
thermophilasig_peptide(1)..(20)CDS(1)..(1350)mat_peptide(61)..(1350)
17atg aag cag tac ctc cag tac ctc gcg gcg acc ctg ccc ctg gtg ggc
48Met Lys Gln Tyr Leu Gln Tyr Leu Ala Ala Thr Leu Pro Leu Val Gly
-20 -15 -10 -5 ctg gcc acg gcc cag cag gcg ggt aac ctg cag acc gag
act cac ccc 96Leu Ala Thr Ala Gln Gln Ala Gly Asn Leu Gln Thr Glu
Thr His Pro -1 1 5 10 agg ctc act tgg tcc aag tgc acg gcc ccg gga
tcc tgc caa cag gtc 144Arg Leu Thr Trp Ser Lys Cys Thr Ala Pro Gly
Ser Cys Gln Gln Val 15 20 25 aac ggc gag gtc gtc atc gac tcc aac
tgg cgc tgg gtg cac gac gag 192Asn Gly Glu Val Val Ile Asp Ser Asn
Trp Arg Trp Val His Asp Glu 30 35 40 aac gcg cag aac tgc tac gac
ggc aac cag tgg acc aac gct tgc agc 240Asn Ala Gln Asn Cys Tyr Asp
Gly Asn Gln Trp Thr Asn Ala Cys Ser 45 50 55 60 tct gcc acc gac tgc
gcc gag aat tgc gcg ctc gag ggt gcc gac tac 288Ser Ala Thr Asp Cys
Ala Glu Asn Cys Ala Leu Glu Gly Ala Asp Tyr 65 70 75 cag ggc acc
tat ggc gcc tcg acc agc ggc aat gcc ctg acg ctc acc 336Gln Gly Thr
Tyr Gly Ala Ser Thr Ser Gly Asn Ala Leu Thr Leu Thr 80 85 90 ttc
gtc act aag cac gag tac ggc acc aac att ggc tcg cgc ctc tac 384Phe
Val Thr Lys His Glu Tyr Gly Thr Asn Ile Gly Ser Arg Leu Tyr 95 100
105 ctc atg aac ggc gcg aac aag tac cag atg ttc acc ctc aag ggc aac
432Leu Met Asn Gly Ala Asn Lys Tyr Gln Met Phe Thr Leu Lys Gly Asn
110 115 120 gag ctg gcc ttc gac gtc gac ctc tcg gcc gtc gag tgc ggc
ctc aac 480Glu Leu Ala Phe Asp Val Asp Leu Ser Ala Val Glu Cys Gly
Leu Asn 125 130 135 140 agc gcc ctc tac ttc gtg gcc atg gag gag gat
ggc ggt gtg tcg agc 528Ser Ala Leu Tyr Phe Val Ala Met Glu Glu Asp
Gly Gly Val Ser Ser 145 150 155 tac ccg acc aac acg gcc ggt gct aag
ttc ggc act ggg tac tgc gac 576Tyr Pro Thr Asn Thr Ala Gly Ala Lys
Phe Gly Thr Gly Tyr Cys Asp 160 165 170 gcc caa tgc gca cgc gac ctc
aag ttc gtc ggc ggc aag ggc aac atc 624Ala Gln Cys Ala Arg Asp Leu
Lys Phe Val Gly Gly Lys Gly Asn Ile 175 180 185 gag ggc tgg aag ccg
tcc acc aac gat gcc aat gcc ggt gtc ggt cct 672Glu Gly Trp Lys Pro
Ser Thr Asn Asp Ala Asn Ala Gly Val Gly Pro 190 195 200 tat ggc ggg
tgc tgc gct gag atc gac gtc tgg gag tcg aac aag tat 720Tyr Gly Gly
Cys Cys Ala Glu Ile Asp Val Trp Glu Ser Asn Lys Tyr 205 210 215 220
gct ttc gct ttc acc ccg cac ggt tgc gag aac cct aaa tac cac gtc
768Ala Phe Ala Phe Thr Pro His Gly Cys Glu Asn Pro Lys Tyr His Val
225 230 235 tgc gag acc acc aac tgc ggt ggc acc tac tcc gag gac cgc
ttc gct 816Cys Glu Thr Thr Asn Cys Gly Gly Thr Tyr Ser Glu Asp Arg
Phe Ala 240 245 250 ggt gac tgc gat gcc aac ggc tgc gac tac aac ccc
tac cgc atg ggc 864Gly Asp Cys Asp Ala Asn Gly Cys Asp Tyr Asn Pro
Tyr Arg Met Gly 255 260 265 aac cag gac ttc tac ggt ccc ggc ttg acg
gtc gat acc agc aag aag 912Asn Gln Asp Phe Tyr Gly Pro Gly Leu Thr
Val Asp Thr Ser Lys Lys 270 275 280 ttc acc gtc gtc agc cag ttc gag
gag aac aag ctc acc cag ttc ttc 960Phe Thr Val Val Ser Gln Phe Glu
Glu Asn Lys Leu Thr Gln Phe Phe 285 290 295 300 gtc cag gac ggc aag
aag att gag atc ccc ggc ccc aag gtc gag ggc 1008Val Gln Asp Gly Lys
Lys Ile Glu Ile Pro Gly Pro Lys Val Glu Gly 305 310 315 atc gat gcg
gac agc gcc gct atc acc cct gag ctg tgc agt gcc ctg 1056Ile Asp Ala
Asp Ser Ala Ala Ile Thr Pro Glu Leu Cys Ser Ala Leu 320 325 330 ttc
aag gcc ttc gat gac cgt gac cgc ttc tcg gag gtt ggc ggc ttc 1104Phe
Lys Ala Phe Asp Asp Arg Asp Arg Phe Ser Glu Val Gly Gly Phe 335 340
345 gat gcc atc aac acg gcc ctc agc act ccc atg gtc ctc gtc atg tcc
1152Asp Ala Ile Asn Thr Ala Leu Ser Thr Pro Met Val Leu Val Met Ser
350 355 360 atc tgg gat gat cac tac gcc aat atg ctc tgg ctc gac tcg
agc tac 1200Ile Trp Asp Asp His Tyr Ala Asn Met Leu Trp Leu Asp Ser
Ser Tyr 365 370 375 380 ccc cct gag aag gct ggc cag cct ggc ggt gac
cgt ggc ccg tgt cct 1248Pro Pro Glu Lys Ala Gly Gln Pro Gly Gly Asp
Arg Gly Pro Cys Pro 385 390 395 cag gac tct ggc gtc ccg gcc gac gtt
gag gct cag tac cct aat gcc 1296Gln Asp Ser Gly Val Pro Ala Asp Val
Glu Ala Gln Tyr Pro Asn Ala 400 405 410 aag gtc atc tgg tcc aac atc
cgc ttc ggc ccc atc ggc tcg act gtc 1344Lys Val Ile Trp Ser Asn Ile
Arg Phe Gly Pro Ile Gly Ser Thr Val 415 420 425 aac gtc taa 1353Asn
Val 430 18450PRTMyceliophthora thermophila 18Met Lys Gln Tyr Leu
Gln Tyr Leu Ala Ala Thr Leu Pro Leu Val Gly -20 -15 -10 -5 Leu Ala
Thr Ala Gln Gln Ala Gly Asn Leu Gln Thr Glu Thr His Pro -1 1 5 10
Arg Leu Thr Trp Ser Lys Cys Thr Ala Pro Gly Ser Cys Gln Gln Val 15
20 25 Asn Gly Glu Val Val Ile Asp Ser Asn Trp Arg Trp Val His Asp
Glu 30 35 40 Asn Ala Gln Asn Cys Tyr Asp Gly Asn Gln Trp Thr Asn
Ala Cys Ser 45 50 55 60 Ser Ala Thr Asp Cys Ala Glu Asn Cys Ala Leu
Glu Gly Ala Asp Tyr 65 70 75 Gln Gly Thr Tyr Gly Ala Ser Thr Ser
Gly Asn Ala Leu Thr Leu Thr 80 85 90 Phe Val Thr Lys His Glu Tyr
Gly Thr Asn Ile Gly Ser Arg Leu Tyr 95 100 105 Leu Met Asn Gly Ala
Asn Lys Tyr Gln Met Phe Thr Leu Lys Gly Asn 110 115 120 Glu Leu Ala
Phe Asp Val Asp Leu Ser Ala Val Glu Cys Gly Leu Asn 125 130 135 140
Ser Ala Leu Tyr Phe Val Ala Met Glu Glu Asp Gly Gly Val Ser Ser 145
150 155 Tyr Pro Thr Asn Thr Ala Gly Ala Lys Phe Gly Thr Gly Tyr Cys
Asp 160 165 170 Ala Gln Cys Ala Arg Asp Leu Lys Phe Val Gly Gly Lys
Gly Asn Ile 175 180 185 Glu Gly Trp Lys Pro Ser Thr Asn Asp Ala Asn
Ala Gly Val Gly Pro 190 195 200 Tyr Gly Gly Cys Cys Ala Glu Ile Asp
Val Trp Glu Ser Asn Lys Tyr 205 210 215 220 Ala Phe Ala Phe Thr Pro
His Gly Cys Glu Asn Pro Lys Tyr His Val 225 230 235 Cys Glu Thr Thr
Asn Cys Gly Gly Thr Tyr Ser Glu Asp Arg Phe Ala 240 245 250 Gly Asp
Cys Asp Ala Asn Gly Cys Asp Tyr Asn Pro Tyr Arg Met Gly 255 260 265
Asn Gln Asp Phe Tyr Gly Pro Gly Leu Thr Val Asp Thr Ser Lys Lys 270
275 280 Phe Thr Val Val Ser Gln Phe Glu Glu Asn Lys Leu Thr Gln Phe
Phe 285 290 295 300 Val Gln Asp Gly Lys Lys Ile Glu Ile Pro Gly Pro
Lys Val Glu Gly 305 310 315 Ile Asp Ala Asp Ser Ala Ala Ile Thr Pro
Glu Leu Cys Ser Ala Leu
320 325 330 Phe Lys Ala Phe Asp Asp Arg Asp Arg Phe Ser Glu Val Gly
Gly Phe 335 340 345 Asp Ala Ile Asn Thr Ala Leu Ser Thr Pro Met Val
Leu Val Met Ser 350 355 360 Ile Trp Asp Asp His Tyr Ala Asn Met Leu
Trp Leu Asp Ser Ser Tyr 365 370 375 380 Pro Pro Glu Lys Ala Gly Gln
Pro Gly Gly Asp Arg Gly Pro Cys Pro 385 390 395 Gln Asp Ser Gly Val
Pro Ala Asp Val Glu Ala Gln Tyr Pro Asn Ala 400 405 410 Lys Val Ile
Trp Ser Asn Ile Arg Phe Gly Pro Ile Gly Ser Thr Val 415 420 425 Asn
Val 430 191590DNAChaetomium
thermophilumsig_peptide(1)..(18)CDS(1)..(1587)mat_peptide(55)..(1587)
19atg atg tac aag aag ttc gcc gct ctc gcc gcc ctc gtg gct ggc gcc
48Met Met Tyr Lys Lys Phe Ala Ala Leu Ala Ala Leu Val Ala Gly Ala
-15 -10 -5 gcc gcc cag cag gct tgc tcc ctc acc act gag acc cac ccc
aga ctc 96Ala Ala Gln Gln Ala Cys Ser Leu Thr Thr Glu Thr His Pro
Arg Leu -1 1 5 10 act tgg aag cgc tgc acc tct ggc ggc aac tgc tcg
acc gtg aac ggc 144Thr Trp Lys Arg Cys Thr Ser Gly Gly Asn Cys Ser
Thr Val Asn Gly 15 20 25 30 gcc gtc acc atc gat gcc aac tgg cgc tgg
act cac acc gtt tcc ggc 192Ala Val Thr Ile Asp Ala Asn Trp Arg Trp
Thr His Thr Val Ser Gly 35 40 45 tcg acc aac tgc tac acc ggc aac
gag tgg gat acc tcc atc tgc tct 240Ser Thr Asn Cys Tyr Thr Gly Asn
Glu Trp Asp Thr Ser Ile Cys Ser 50 55 60 gat ggc aag agc tgc gcc
cag acc tgc tgc gtc gac ggc gct gac tac 288Asp Gly Lys Ser Cys Ala
Gln Thr Cys Cys Val Asp Gly Ala Asp Tyr 65 70 75 tct tcg acc tat
ggt atc acc acc agc ggt gac tcc ctg aac ctc aag 336Ser Ser Thr Tyr
Gly Ile Thr Thr Ser Gly Asp Ser Leu Asn Leu Lys 80 85 90 ttc gtc
acc aag cac cag tac ggc acc aat gtc ggc tct cgt gtc tac 384Phe Val
Thr Lys His Gln Tyr Gly Thr Asn Val Gly Ser Arg Val Tyr 95 100 105
110 ctg atg gag aac gac acc aag tac cag atg ttc gag ctc ctc ggc aac
432Leu Met Glu Asn Asp Thr Lys Tyr Gln Met Phe Glu Leu Leu Gly Asn
115 120 125 gag ttc acc ttc gat gtc gat gtc tct aac ctg ggc tgc ggt
ctc aac 480Glu Phe Thr Phe Asp Val Asp Val Ser Asn Leu Gly Cys Gly
Leu Asn 130 135 140 ggt gcc ctc tac ttc gtc tcc atg gac gct gat ggt
ggt atg agc aag 528Gly Ala Leu Tyr Phe Val Ser Met Asp Ala Asp Gly
Gly Met Ser Lys 145 150 155 tac tct ggc aac aag gct ggc gcc aag tac
ggt acc ggc tac tgc gat 576Tyr Ser Gly Asn Lys Ala Gly Ala Lys Tyr
Gly Thr Gly Tyr Cys Asp 160 165 170 gct cag tgc ccg cgc gac ctt aag
ttc atc aac ggc gag gcc aac att 624Ala Gln Cys Pro Arg Asp Leu Lys
Phe Ile Asn Gly Glu Ala Asn Ile 175 180 185 190 gag aac tgg acc cct
tcg acc aat gat gcc aac gcc ggt ttc ggc cgc 672Glu Asn Trp Thr Pro
Ser Thr Asn Asp Ala Asn Ala Gly Phe Gly Arg 195 200 205 tat ggc agc
tgc tgc tct gag atg gat atc tgg gag gcc aac aac atg 720Tyr Gly Ser
Cys Cys Ser Glu Met Asp Ile Trp Glu Ala Asn Asn Met 210 215 220 gct
act gcc ttc act cct cac cct tgc acc att atc ggc cag agc cgc 768Ala
Thr Ala Phe Thr Pro His Pro Cys Thr Ile Ile Gly Gln Ser Arg 225 230
235 tgc gag ggc aac agc tgc ggt ggc acc tac agc tct gag cgc tat gct
816Cys Glu Gly Asn Ser Cys Gly Gly Thr Tyr Ser Ser Glu Arg Tyr Ala
240 245 250 ggt gtt tgc gat cct gat ggc tgc gac ttc aac gcc tac cgc
cag ggc 864Gly Val Cys Asp Pro Asp Gly Cys Asp Phe Asn Ala Tyr Arg
Gln Gly 255 260 265 270 gac aag acc ttc tac ggc aag ggc atg acc gtc
gac acc acc aag aag 912Asp Lys Thr Phe Tyr Gly Lys Gly Met Thr Val
Asp Thr Thr Lys Lys 275 280 285 atg acc gtc gtc acc cag ttc cac aag
aac tcg gct ggc gtc ctc agc 960Met Thr Val Val Thr Gln Phe His Lys
Asn Ser Ala Gly Val Leu Ser 290 295 300 gag atc aag cgc ttc tac gtt
cag gac ggc aag atc att gcc aac gcc 1008Glu Ile Lys Arg Phe Tyr Val
Gln Asp Gly Lys Ile Ile Ala Asn Ala 305 310 315 gag tcc aag atc ccc
ggc aac ccc ggc aac tcc atc acc cag gag tgg 1056Glu Ser Lys Ile Pro
Gly Asn Pro Gly Asn Ser Ile Thr Gln Glu Trp 320 325 330 tgc gat gcc
cag aag gtc gcc ttc ggt gac atc gat gac ttc aac cgc 1104Cys Asp Ala
Gln Lys Val Ala Phe Gly Asp Ile Asp Asp Phe Asn Arg 335 340 345 350
aag ggc ggt atg gct cag atg agc aag gcc ctc gag ggc cct atg gtc
1152Lys Gly Gly Met Ala Gln Met Ser Lys Ala Leu Glu Gly Pro Met Val
355 360 365 ctg gtc atg tcc gtc tgg gat gac cac tac gcc aac atg ctc
tgg ctc 1200Leu Val Met Ser Val Trp Asp Asp His Tyr Ala Asn Met Leu
Trp Leu 370 375 380 gac tcg acc tac ccc atc gac aag gcc ggc acc ccc
ggc gcc gag cgc 1248Asp Ser Thr Tyr Pro Ile Asp Lys Ala Gly Thr Pro
Gly Ala Glu Arg 385 390 395 ggt gct tgc ccg acc acc tcc ggt gtc cct
gcc gag att gag gcc cag 1296Gly Ala Cys Pro Thr Thr Ser Gly Val Pro
Ala Glu Ile Glu Ala Gln 400 405 410 gtc ccc aac agc aac gtc atc ttc
tcc aac atc cgc ttc ggc ccc atc 1344Val Pro Asn Ser Asn Val Ile Phe
Ser Asn Ile Arg Phe Gly Pro Ile 415 420 425 430 ggc tcg acc gtc cct
ggc ctc gac ggc agc act ccc agc aac ccg acc 1392Gly Ser Thr Val Pro
Gly Leu Asp Gly Ser Thr Pro Ser Asn Pro Thr 435 440 445 gcc acc gtt
gct cct ccc act tct acc acc agc gtg aga agc agc act 1440Ala Thr Val
Ala Pro Pro Thr Ser Thr Thr Ser Val Arg Ser Ser Thr 450 455 460 act
cag att tcc acc ccg act agc cag ccc ggc ggc tgc acc acc cag 1488Thr
Gln Ile Ser Thr Pro Thr Ser Gln Pro Gly Gly Cys Thr Thr Gln 465 470
475 aag tgg ggc cag tgc ggt ggt atc ggc tac acc ggc tgc act aac tgc
1536Lys Trp Gly Gln Cys Gly Gly Ile Gly Tyr Thr Gly Cys Thr Asn Cys
480 485 490 gtt gct ggc act acc tgc act gag ctc aac ccc tgg tac agc
cag tgc 1584Val Ala Gly Thr Thr Cys Thr Glu Leu Asn Pro Trp Tyr Ser
Gln Cys 495 500 505 510 ctg taa 1590Leu 20529PRTChaetomium
thermophilum 20Met Met Tyr Lys Lys Phe Ala Ala Leu Ala Ala Leu Val
Ala Gly Ala -15 -10 -5 Ala Ala Gln Gln Ala Cys Ser Leu Thr Thr Glu
Thr His Pro Arg Leu -1 1 5 10 Thr Trp Lys Arg Cys Thr Ser Gly Gly
Asn Cys Ser Thr Val Asn Gly 15 20 25 30 Ala Val Thr Ile Asp Ala Asn
Trp Arg Trp Thr His Thr Val Ser Gly 35 40 45 Ser Thr Asn Cys Tyr
Thr Gly Asn Glu Trp Asp Thr Ser Ile Cys Ser 50 55 60 Asp Gly Lys
Ser Cys Ala Gln Thr Cys Cys Val Asp Gly Ala Asp Tyr 65 70 75 Ser
Ser Thr Tyr Gly Ile Thr Thr Ser Gly Asp Ser Leu Asn Leu Lys 80 85
90 Phe Val Thr Lys His Gln Tyr Gly Thr Asn Val Gly Ser Arg Val Tyr
95 100 105 110 Leu Met Glu Asn Asp Thr Lys Tyr Gln Met Phe Glu Leu
Leu Gly Asn 115 120 125 Glu Phe Thr Phe Asp Val Asp Val Ser Asn Leu
Gly Cys Gly Leu Asn 130 135 140 Gly Ala Leu Tyr Phe Val Ser Met Asp
Ala Asp Gly Gly Met Ser Lys 145 150 155 Tyr Ser Gly Asn Lys Ala Gly
Ala Lys Tyr Gly Thr Gly Tyr Cys Asp 160 165 170 Ala Gln Cys Pro Arg
Asp Leu Lys Phe Ile Asn Gly Glu Ala Asn Ile 175 180 185 190 Glu Asn
Trp Thr Pro Ser Thr Asn Asp Ala Asn Ala Gly Phe Gly Arg 195 200 205
Tyr Gly Ser Cys Cys Ser Glu Met Asp Ile Trp Glu Ala Asn Asn Met 210
215 220 Ala Thr Ala Phe Thr Pro His Pro Cys Thr Ile Ile Gly Gln Ser
Arg 225 230 235 Cys Glu Gly Asn Ser Cys Gly Gly Thr Tyr Ser Ser Glu
Arg Tyr Ala 240 245 250 Gly Val Cys Asp Pro Asp Gly Cys Asp Phe Asn
Ala Tyr Arg Gln Gly 255 260 265 270 Asp Lys Thr Phe Tyr Gly Lys Gly
Met Thr Val Asp Thr Thr Lys Lys 275 280 285 Met Thr Val Val Thr Gln
Phe His Lys Asn Ser Ala Gly Val Leu Ser 290 295 300 Glu Ile Lys Arg
Phe Tyr Val Gln Asp Gly Lys Ile Ile Ala Asn Ala 305 310 315 Glu Ser
Lys Ile Pro Gly Asn Pro Gly Asn Ser Ile Thr Gln Glu Trp 320 325 330
Cys Asp Ala Gln Lys Val Ala Phe Gly Asp Ile Asp Asp Phe Asn Arg 335
340 345 350 Lys Gly Gly Met Ala Gln Met Ser Lys Ala Leu Glu Gly Pro
Met Val 355 360 365 Leu Val Met Ser Val Trp Asp Asp His Tyr Ala Asn
Met Leu Trp Leu 370 375 380 Asp Ser Thr Tyr Pro Ile Asp Lys Ala Gly
Thr Pro Gly Ala Glu Arg 385 390 395 Gly Ala Cys Pro Thr Thr Ser Gly
Val Pro Ala Glu Ile Glu Ala Gln 400 405 410 Val Pro Asn Ser Asn Val
Ile Phe Ser Asn Ile Arg Phe Gly Pro Ile 415 420 425 430 Gly Ser Thr
Val Pro Gly Leu Asp Gly Ser Thr Pro Ser Asn Pro Thr 435 440 445 Ala
Thr Val Ala Pro Pro Thr Ser Thr Thr Ser Val Arg Ser Ser Thr 450 455
460 Thr Gln Ile Ser Thr Pro Thr Ser Gln Pro Gly Gly Cys Thr Thr Gln
465 470 475 Lys Trp Gly Gln Cys Gly Gly Ile Gly Tyr Thr Gly Cys Thr
Asn Cys 480 485 490 Val Ala Gly Thr Thr Cys Thr Glu Leu Asn Pro Trp
Tyr Ser Gln Cys 495 500 505 510 Leu 211578DNAHumicola
griseasig_peptide(1)..(18)CDS(1)..(1575)mat_peptide(55)..(1575)
21atg cgt acc gcc aag ttc gcc acc ctc gcc gcc ctt gtg gcc tcg gcc
48Met Arg Thr Ala Lys Phe Ala Thr Leu Ala Ala Leu Val Ala Ser Ala
-15 -10 -5 gcc gcc cag cag gcg tgc agt ctc acc acc gag agg cac cct
tcc ctc 96Ala Ala Gln Gln Ala Cys Ser Leu Thr Thr Glu Arg His Pro
Ser Leu -1 1 5 10 tct tgg aag aag tgc acc gcc ggc ggc cag tgc cag
acc gtc cag gct 144Ser Trp Lys Lys Cys Thr Ala Gly Gly Gln Cys Gln
Thr Val Gln Ala 15 20 25 30 tcc atc act ctc gac tcc aac tgg cgc tgg
act cac cag gtg tct ggc 192Ser Ile Thr Leu Asp Ser Asn Trp Arg Trp
Thr His Gln Val Ser Gly 35 40 45 tcc acc aac tgc tac acg ggc aac
aag tgg gat act agc atc tgc act 240Ser Thr Asn Cys Tyr Thr Gly Asn
Lys Trp Asp Thr Ser Ile Cys Thr 50 55 60 gat gcc aag tcg tgc gct
cag aac tgc tgc gtc gat ggt gcc gac tac 288Asp Ala Lys Ser Cys Ala
Gln Asn Cys Cys Val Asp Gly Ala Asp Tyr 65 70 75 acc agc acc tat
ggc atc acc acc aac ggt gat tcc ctg agc ctc aag 336Thr Ser Thr Tyr
Gly Ile Thr Thr Asn Gly Asp Ser Leu Ser Leu Lys 80 85 90 ttc gtc
acc aag ggc cag cac tcg acc aac gtc ggc tcg cgt acc tac 384Phe Val
Thr Lys Gly Gln His Ser Thr Asn Val Gly Ser Arg Thr Tyr 95 100 105
110 ctg atg gac ggc gag gac aag tat cag acc ttc gag ctc ctc ggc aac
432Leu Met Asp Gly Glu Asp Lys Tyr Gln Thr Phe Glu Leu Leu Gly Asn
115 120 125 gag ttc acc ttc gat gtc gat gtc tcc aac atc ggc tgc ggt
ctc aac 480Glu Phe Thr Phe Asp Val Asp Val Ser Asn Ile Gly Cys Gly
Leu Asn 130 135 140 ggc gcc ctg tac ttc gtc tcc atg gac gcc gat ggt
ggt ctc agc cgc 528Gly Ala Leu Tyr Phe Val Ser Met Asp Ala Asp Gly
Gly Leu Ser Arg 145 150 155 tat cct ggc aac aag gct ggt gcc aag tac
ggt acc ggc tac tgc gat 576Tyr Pro Gly Asn Lys Ala Gly Ala Lys Tyr
Gly Thr Gly Tyr Cys Asp 160 165 170 gct cag tgc ccc cgt gac atc aag
ttc atc aac ggc gag gcc aac att 624Ala Gln Cys Pro Arg Asp Ile Lys
Phe Ile Asn Gly Glu Ala Asn Ile 175 180 185 190 gag ggc tgg acc ggc
tcc acc aac gac ccc aac gcc ggc gcg ggc cgc 672Glu Gly Trp Thr Gly
Ser Thr Asn Asp Pro Asn Ala Gly Ala Gly Arg 195 200 205 tat ggt acc
tgc tgc tct gag atg gat atc tgg gaa gcc aac aac atg 720Tyr Gly Thr
Cys Cys Ser Glu Met Asp Ile Trp Glu Ala Asn Asn Met 210 215 220 gct
act gcc ttc act cct cac cct tgc acc atc att ggc cag agc cgc 768Ala
Thr Ala Phe Thr Pro His Pro Cys Thr Ile Ile Gly Gln Ser Arg 225 230
235 tgc gag ggc gac tcg tgc ggt ggc acc tac agc aac gag cgc tac gcc
816Cys Glu Gly Asp Ser Cys Gly Gly Thr Tyr Ser Asn Glu Arg Tyr Ala
240 245 250 ggc gtc tgc gac ccc gat ggc tgc gac ttc aac tcg tac cgc
cag ggc 864Gly Val Cys Asp Pro Asp Gly Cys Asp Phe Asn Ser Tyr Arg
Gln Gly 255 260 265 270 aat aag acc ttc tac ggc aag ggc atg acc gtc
gac acc acc aag aag 912Asn Lys Thr Phe Tyr Gly Lys Gly Met Thr Val
Asp Thr Thr Lys Lys 275 280 285 atc act gtc gtc acc cag ttc ctc aag
gat gcc aac ggc gat ctc ggc 960Ile Thr Val Val Thr Gln Phe Leu Lys
Asp Ala Asn Gly Asp Leu Gly 290 295 300 gag gtc aag cgc ttc tac gtc
cag gat ggc aag atc atc ccc aac tcc 1008Glu Val Lys Arg Phe Tyr Val
Gln Asp Gly Lys Ile Ile Pro Asn Ser 305 310 315 gag tcc acc atc ccc
ggc gtc gag ggc aat tcc atc acc cag gac tgg 1056Glu Ser Thr Ile Pro
Gly Val Glu Gly Asn Ser Ile Thr Gln Asp Trp 320 325 330 tgc gac cgc
cag aag gtt gcc ttt ggc gac att gac gac ttc aac cgc 1104Cys Asp Arg
Gln Lys Val Ala Phe Gly Asp Ile Asp Asp Phe Asn Arg 335 340 345 350
aag ggc ggc atg aag cag atg ggc aag gcc ctc gcc ggc ccc atg gtc
1152Lys Gly Gly Met Lys Gln Met Gly Lys Ala Leu Ala Gly Pro Met Val
355 360 365 ctg gtc atg tcc atc tgg gat gac cac gcc tcc aac atg ctc
tgg ctc 1200Leu Val Met Ser Ile Trp Asp Asp His Ala Ser Asn Met Leu
Trp Leu 370 375 380 gac tcg acc ttc cct gtc gat gcc gct ggc aag ccc
ggc gcc gag cgc 1248Asp Ser Thr Phe Pro Val Asp Ala Ala Gly Lys Pro
Gly Ala Glu Arg 385 390 395
ggt gcc tgc ccg acc acc tcg ggt gtc cct gct gag gtt gag gcc gag
1296Gly Ala Cys Pro Thr Thr Ser Gly Val Pro Ala Glu Val Glu Ala Glu
400 405 410 gcc ccc aac agc aac gtc gtc ttc tcc aac atc cgc ttc ggc
ccc atc 1344Ala Pro Asn Ser Asn Val Val Phe Ser Asn Ile Arg Phe Gly
Pro Ile 415 420 425 430 ggc tcg acc gtt gct ggt ctc ccc ggc gcg ggc
aac ggc ggc aac aac 1392Gly Ser Thr Val Ala Gly Leu Pro Gly Ala Gly
Asn Gly Gly Asn Asn 435 440 445 ggc ggc aac ccc ccg ccc ccc acc acc
acc acc tcc tcg gct ccg gcc 1440Gly Gly Asn Pro Pro Pro Pro Thr Thr
Thr Thr Ser Ser Ala Pro Ala 450 455 460 acc acc acc acc gcc agc gct
ggc ccc aag gct ggc cac tgg cag cag 1488Thr Thr Thr Thr Ala Ser Ala
Gly Pro Lys Ala Gly His Trp Gln Gln 465 470 475 tgc ggc ggc atc ggc
ttc act ggc ccg acc cag tgc gag gag ccc tac 1536Cys Gly Gly Ile Gly
Phe Thr Gly Pro Thr Gln Cys Glu Glu Pro Tyr 480 485 490 act tgc acc
aag ctc aac gac tgg tac tct cag tgc ctg taa 1578Thr Cys Thr Lys Leu
Asn Asp Trp Tyr Ser Gln Cys Leu 495 500 505 22525PRTHumicola grisea
22Met Arg Thr Ala Lys Phe Ala Thr Leu Ala Ala Leu Val Ala Ser Ala
-15 -10 -5 Ala Ala Gln Gln Ala Cys Ser Leu Thr Thr Glu Arg His Pro
Ser Leu -1 1 5 10 Ser Trp Lys Lys Cys Thr Ala Gly Gly Gln Cys Gln
Thr Val Gln Ala 15 20 25 30 Ser Ile Thr Leu Asp Ser Asn Trp Arg Trp
Thr His Gln Val Ser Gly 35 40 45 Ser Thr Asn Cys Tyr Thr Gly Asn
Lys Trp Asp Thr Ser Ile Cys Thr 50 55 60 Asp Ala Lys Ser Cys Ala
Gln Asn Cys Cys Val Asp Gly Ala Asp Tyr 65 70 75 Thr Ser Thr Tyr
Gly Ile Thr Thr Asn Gly Asp Ser Leu Ser Leu Lys 80 85 90 Phe Val
Thr Lys Gly Gln His Ser Thr Asn Val Gly Ser Arg Thr Tyr 95 100 105
110 Leu Met Asp Gly Glu Asp Lys Tyr Gln Thr Phe Glu Leu Leu Gly Asn
115 120 125 Glu Phe Thr Phe Asp Val Asp Val Ser Asn Ile Gly Cys Gly
Leu Asn 130 135 140 Gly Ala Leu Tyr Phe Val Ser Met Asp Ala Asp Gly
Gly Leu Ser Arg 145 150 155 Tyr Pro Gly Asn Lys Ala Gly Ala Lys Tyr
Gly Thr Gly Tyr Cys Asp 160 165 170 Ala Gln Cys Pro Arg Asp Ile Lys
Phe Ile Asn Gly Glu Ala Asn Ile 175 180 185 190 Glu Gly Trp Thr Gly
Ser Thr Asn Asp Pro Asn Ala Gly Ala Gly Arg 195 200 205 Tyr Gly Thr
Cys Cys Ser Glu Met Asp Ile Trp Glu Ala Asn Asn Met 210 215 220 Ala
Thr Ala Phe Thr Pro His Pro Cys Thr Ile Ile Gly Gln Ser Arg 225 230
235 Cys Glu Gly Asp Ser Cys Gly Gly Thr Tyr Ser Asn Glu Arg Tyr Ala
240 245 250 Gly Val Cys Asp Pro Asp Gly Cys Asp Phe Asn Ser Tyr Arg
Gln Gly 255 260 265 270 Asn Lys Thr Phe Tyr Gly Lys Gly Met Thr Val
Asp Thr Thr Lys Lys 275 280 285 Ile Thr Val Val Thr Gln Phe Leu Lys
Asp Ala Asn Gly Asp Leu Gly 290 295 300 Glu Val Lys Arg Phe Tyr Val
Gln Asp Gly Lys Ile Ile Pro Asn Ser 305 310 315 Glu Ser Thr Ile Pro
Gly Val Glu Gly Asn Ser Ile Thr Gln Asp Trp 320 325 330 Cys Asp Arg
Gln Lys Val Ala Phe Gly Asp Ile Asp Asp Phe Asn Arg 335 340 345 350
Lys Gly Gly Met Lys Gln Met Gly Lys Ala Leu Ala Gly Pro Met Val 355
360 365 Leu Val Met Ser Ile Trp Asp Asp His Ala Ser Asn Met Leu Trp
Leu 370 375 380 Asp Ser Thr Phe Pro Val Asp Ala Ala Gly Lys Pro Gly
Ala Glu Arg 385 390 395 Gly Ala Cys Pro Thr Thr Ser Gly Val Pro Ala
Glu Val Glu Ala Glu 400 405 410 Ala Pro Asn Ser Asn Val Val Phe Ser
Asn Ile Arg Phe Gly Pro Ile 415 420 425 430 Gly Ser Thr Val Ala Gly
Leu Pro Gly Ala Gly Asn Gly Gly Asn Asn 435 440 445 Gly Gly Asn Pro
Pro Pro Pro Thr Thr Thr Thr Ser Ser Ala Pro Ala 450 455 460 Thr Thr
Thr Thr Ala Ser Ala Gly Pro Lys Ala Gly His Trp Gln Gln 465 470 475
Cys Gly Gly Ile Gly Phe Thr Gly Pro Thr Gln Cys Glu Glu Pro Tyr 480
485 490 Thr Cys Thr Lys Leu Asn Asp Trp Tyr Ser Gln Cys Leu 495 500
505 231560DNARasamsonia
emersoniisig_peptide(1)..(18)CDS(1)..(1557)mat_peptide(55)..(1557)
23atg ttg cga agg gcc ttg ttg ctc tcg tcc tcc gca atc ttg gcg gtc
48Met Leu Arg Arg Ala Leu Leu Leu Ser Ser Ser Ala Ile Leu Ala Val
-15 -10 -5 aag gca cag cag gca ggc acc gca acc gca gag aac cat cct
ccg ctc 96Lys Ala Gln Gln Ala Gly Thr Ala Thr Ala Glu Asn His Pro
Pro Leu -1 1 5 10 act tgg cag gaa tgt aca gca cct ggc tcc tgt aca
acc cag aac gga 144Thr Trp Gln Glu Cys Thr Ala Pro Gly Ser Cys Thr
Thr Gln Asn Gly 15 20 25 30 gcg gtc gtg ctc gat gcg aac tgg cgc tgg
gtg cac gat gtc aac gga 192Ala Val Val Leu Asp Ala Asn Trp Arg Trp
Val His Asp Val Asn Gly 35 40 45 tac aca aac tgt tat aca ggt aac
acg tgg aac cct acg tat tgt ccc 240Tyr Thr Asn Cys Tyr Thr Gly Asn
Thr Trp Asn Pro Thr Tyr Cys Pro 50 55 60 gac gac gaa acg tgt gcc
cag aac tgt gcg ttg gat gga gca gac tac 288Asp Asp Glu Thr Cys Ala
Gln Asn Cys Ala Leu Asp Gly Ala Asp Tyr 65 70 75 gag gga acg tat
ggc gtg acc tcg tcc ggc tcc tcc ttg aag ctc aac 336Glu Gly Thr Tyr
Gly Val Thr Ser Ser Gly Ser Ser Leu Lys Leu Asn 80 85 90 ttc gtc
acg ggc tcg aac gtc ggc tcc cgc ttg tac ctc ctc cag gac 384Phe Val
Thr Gly Ser Asn Val Gly Ser Arg Leu Tyr Leu Leu Gln Asp 95 100 105
110 gac tcg acc tac cag atc ttc aag ctc ctc aac agg gag ttc acc ttc
432Asp Ser Thr Tyr Gln Ile Phe Lys Leu Leu Asn Arg Glu Phe Thr Phe
115 120 125 gac gtc gat gtc tcc aac ttg ccc tgt ggt ctc aac gga gcc
ttg tac 480Asp Val Asp Val Ser Asn Leu Pro Cys Gly Leu Asn Gly Ala
Leu Tyr 130 135 140 ttc gtc gcg atg gat gca gac gga ggt gtc tcg aag
tac ccc aac aac 528Phe Val Ala Met Asp Ala Asp Gly Gly Val Ser Lys
Tyr Pro Asn Asn 145 150 155 aag gca ggt gcc aag tat ggt act ggc tac
tgt gat tcg cag tgt cct 576Lys Ala Gly Ala Lys Tyr Gly Thr Gly Tyr
Cys Asp Ser Gln Cys Pro 160 165 170 cgc gat ctc aag ttc att gac ggt
gag gcg aac gtg gaa gga tgg cag 624Arg Asp Leu Lys Phe Ile Asp Gly
Glu Ala Asn Val Glu Gly Trp Gln 175 180 185 190 ccc tcg tcc aac aac
gcg aac act ggc atc ggt gat cac ggt tcg tgt 672Pro Ser Ser Asn Asn
Ala Asn Thr Gly Ile Gly Asp His Gly Ser Cys 195 200 205 tgt gcc gag
atg gac gtc tgg gaa gcc aac tcc atc tcg aac gcg gtc 720Cys Ala Glu
Met Asp Val Trp Glu Ala Asn Ser Ile Ser Asn Ala Val 210 215 220 aca
ccg cac ccg tgt gat act cct ggc cag act atg tgt tcc gga gat 768Thr
Pro His Pro Cys Asp Thr Pro Gly Gln Thr Met Cys Ser Gly Asp 225 230
235 gat tgt gga ggc acc tat tcg aac gac cgg tat gca ggc acg tgt gac
816Asp Cys Gly Gly Thr Tyr Ser Asn Asp Arg Tyr Ala Gly Thr Cys Asp
240 245 250 ccg gat ggc tgt gac ttc aac ccg tac cgc atg ggc aac acc
tcc ttc 864Pro Asp Gly Cys Asp Phe Asn Pro Tyr Arg Met Gly Asn Thr
Ser Phe 255 260 265 270 tat gga ccg ggt aag atc atc gat aca act aag
ccc ttc acc gtc gtc 912Tyr Gly Pro Gly Lys Ile Ile Asp Thr Thr Lys
Pro Phe Thr Val Val 275 280 285 acg cag ttc ctc aca gat gac ggc acg
gac aca ggt act ttg tcg gag 960Thr Gln Phe Leu Thr Asp Asp Gly Thr
Asp Thr Gly Thr Leu Ser Glu 290 295 300 atc aaa cgc ttc tac gtc cag
aac gga aac gtc atc ccc cag ccg aac 1008Ile Lys Arg Phe Tyr Val Gln
Asn Gly Asn Val Ile Pro Gln Pro Asn 305 310 315 tcc gac att tcg gga
gtc aca ggc aac tcg att acg acc gag ttc tgt 1056Ser Asp Ile Ser Gly
Val Thr Gly Asn Ser Ile Thr Thr Glu Phe Cys 320 325 330 aca gcc cag
aaa cag gca ttc ggt gac acg gat gat ttc tcc cag cac 1104Thr Ala Gln
Lys Gln Ala Phe Gly Asp Thr Asp Asp Phe Ser Gln His 335 340 345 350
gga gga ttg gcg aaa atg gga gcc gca atg cag cag gga atg gtc ctc
1152Gly Gly Leu Ala Lys Met Gly Ala Ala Met Gln Gln Gly Met Val Leu
355 360 365 gtg atg tcg ctc tgg gac gac tat gca gcc cag atg ttg tgg
ctc gac 1200Val Met Ser Leu Trp Asp Asp Tyr Ala Ala Gln Met Leu Trp
Leu Asp 370 375 380 tcg gac tac ccc aca gac gcc gat ccc acg aca ccc
ggt atc gca cga 1248Ser Asp Tyr Pro Thr Asp Ala Asp Pro Thr Thr Pro
Gly Ile Ala Arg 385 390 395 ggc act tgt ccg aca gat tcc gga gtc ccg
tcg gac gtc gag tcc cag 1296Gly Thr Cys Pro Thr Asp Ser Gly Val Pro
Ser Asp Val Glu Ser Gln 400 405 410 tcc ccc aac tcg tac gtc acc tat
tcg aac atc aaa ttc ggt ccc atc 1344Ser Pro Asn Ser Tyr Val Thr Tyr
Ser Asn Ile Lys Phe Gly Pro Ile 415 420 425 430 aac tcg aca ttc aca
gcc tcg ggt gga aac cct cct ggc gga aac cct 1392Asn Ser Thr Phe Thr
Ala Ser Gly Gly Asn Pro Pro Gly Gly Asn Pro 435 440 445 cct ggc aca
act aca aca cga cgg cct gcg act aca acg ggt tcg tcc 1440Pro Gly Thr
Thr Thr Thr Arg Arg Pro Ala Thr Thr Thr Gly Ser Ser 450 455 460 cct
gga ccg acc cag tcc cac tac gga cag tgt gga ggc atc ggt tat 1488Pro
Gly Pro Thr Gln Ser His Tyr Gly Gln Cys Gly Gly Ile Gly Tyr 465 470
475 tcc ggt ccg acc gtc tgt gcg tcc ggc aca acc tgt cag gtc ttg aac
1536Ser Gly Pro Thr Val Cys Ala Ser Gly Thr Thr Cys Gln Val Leu Asn
480 485 490 cct tac tat tcg cag tgt ctc taa 1560Pro Tyr Tyr Ser Gln
Cys Leu 495 500 24519PRTRasamsonia emersonii 24Met Leu Arg Arg Ala
Leu Leu Leu Ser Ser Ser Ala Ile Leu Ala Val -15 -10 -5 Lys Ala Gln
Gln Ala Gly Thr Ala Thr Ala Glu Asn His Pro Pro Leu -1 1 5 10 Thr
Trp Gln Glu Cys Thr Ala Pro Gly Ser Cys Thr Thr Gln Asn Gly 15 20
25 30 Ala Val Val Leu Asp Ala Asn Trp Arg Trp Val His Asp Val Asn
Gly 35 40 45 Tyr Thr Asn Cys Tyr Thr Gly Asn Thr Trp Asn Pro Thr
Tyr Cys Pro 50 55 60 Asp Asp Glu Thr Cys Ala Gln Asn Cys Ala Leu
Asp Gly Ala Asp Tyr 65 70 75 Glu Gly Thr Tyr Gly Val Thr Ser Ser
Gly Ser Ser Leu Lys Leu Asn 80 85 90 Phe Val Thr Gly Ser Asn Val
Gly Ser Arg Leu Tyr Leu Leu Gln Asp 95 100 105 110 Asp Ser Thr Tyr
Gln Ile Phe Lys Leu Leu Asn Arg Glu Phe Thr Phe 115 120 125 Asp Val
Asp Val Ser Asn Leu Pro Cys Gly Leu Asn Gly Ala Leu Tyr 130 135 140
Phe Val Ala Met Asp Ala Asp Gly Gly Val Ser Lys Tyr Pro Asn Asn 145
150 155 Lys Ala Gly Ala Lys Tyr Gly Thr Gly Tyr Cys Asp Ser Gln Cys
Pro 160 165 170 Arg Asp Leu Lys Phe Ile Asp Gly Glu Ala Asn Val Glu
Gly Trp Gln 175 180 185 190 Pro Ser Ser Asn Asn Ala Asn Thr Gly Ile
Gly Asp His Gly Ser Cys 195 200 205 Cys Ala Glu Met Asp Val Trp Glu
Ala Asn Ser Ile Ser Asn Ala Val 210 215 220 Thr Pro His Pro Cys Asp
Thr Pro Gly Gln Thr Met Cys Ser Gly Asp 225 230 235 Asp Cys Gly Gly
Thr Tyr Ser Asn Asp Arg Tyr Ala Gly Thr Cys Asp 240 245 250 Pro Asp
Gly Cys Asp Phe Asn Pro Tyr Arg Met Gly Asn Thr Ser Phe 255 260 265
270 Tyr Gly Pro Gly Lys Ile Ile Asp Thr Thr Lys Pro Phe Thr Val Val
275 280 285 Thr Gln Phe Leu Thr Asp Asp Gly Thr Asp Thr Gly Thr Leu
Ser Glu 290 295 300 Ile Lys Arg Phe Tyr Val Gln Asn Gly Asn Val Ile
Pro Gln Pro Asn 305 310 315 Ser Asp Ile Ser Gly Val Thr Gly Asn Ser
Ile Thr Thr Glu Phe Cys 320 325 330 Thr Ala Gln Lys Gln Ala Phe Gly
Asp Thr Asp Asp Phe Ser Gln His 335 340 345 350 Gly Gly Leu Ala Lys
Met Gly Ala Ala Met Gln Gln Gly Met Val Leu 355 360 365 Val Met Ser
Leu Trp Asp Asp Tyr Ala Ala Gln Met Leu Trp Leu Asp 370 375 380 Ser
Asp Tyr Pro Thr Asp Ala Asp Pro Thr Thr Pro Gly Ile Ala Arg 385 390
395 Gly Thr Cys Pro Thr Asp Ser Gly Val Pro Ser Asp Val Glu Ser Gln
400 405 410 Ser Pro Asn Ser Tyr Val Thr Tyr Ser Asn Ile Lys Phe Gly
Pro Ile 415 420 425 430 Asn Ser Thr Phe Thr Ala Ser Gly Gly Asn Pro
Pro Gly Gly Asn Pro 435 440 445 Pro Gly Thr Thr Thr Thr Arg Arg Pro
Ala Thr Thr Thr Gly Ser Ser 450 455 460 Pro Gly Pro Thr Gln Ser His
Tyr Gly Gln Cys Gly Gly Ile Gly Tyr 465 470 475 Ser Gly Pro Thr Val
Cys Ala Ser Gly Thr Thr Cys Gln Val Leu Asn 480 485 490 Pro Tyr Tyr
Ser Gln Cys Leu 495 500 251599DNAAspergillus
fumigatussig_peptide(1)..(26)CDS(1)..(1596)mat_peptide(79)..(1596)
25atg ttg gcc tcc acg ttc tcc tat cgc atg tac aaa aca gcg ctc atc
48Met Leu Ala Ser Thr Phe Ser Tyr Arg Met Tyr Lys Thr Ala Leu Ile
-25 -20 -15 ttg gca gcc ctc ttg ggc tcg gga cag gca cag cag gtc gga
acc tcg 96Leu Ala Ala Leu Leu Gly Ser Gly Gln Ala Gln Gln Val Gly
Thr Ser -10 -5 -1 1 5 cag gcc gag gtc cat cct tcc atg acg tgg cag
tcg tgt aca gcg ggt 144Gln Ala Glu Val His Pro Ser Met Thr Trp Gln
Ser Cys Thr Ala Gly 10 15 20 ggt tcg tgt acc aca aac aac ggt aaa
gtc gtg atc gat gca aac tgg 192Gly Ser Cys Thr Thr Asn Asn Gly Lys
Val Val Ile Asp Ala Asn Trp 25 30 35 agg tgg gtg cac aag act ggc
gac tac acc aac tgt tac aca ggc aac 240Arg Trp Val His Lys Thr Gly
Asp Tyr Thr Asn Cys Tyr Thr Gly Asn 40
45 50 aca tgg gat aca acc atc tgt ccc gac gat gcc act tgt gca tcc
aac 288Thr Trp Asp Thr Thr Ile Cys Pro Asp Asp Ala Thr Cys Ala Ser
Asn 55 60 65 70 tgt gca ctc gag ggt gcc aac tat gag tcg acg tac gga
gtg acc gcc 336Cys Ala Leu Glu Gly Ala Asn Tyr Glu Ser Thr Tyr Gly
Val Thr Ala 75 80 85 tcc gga aac tcg ctc agg ctc aac ttc gtc aca
act tcc cag cag aag 384Ser Gly Asn Ser Leu Arg Leu Asn Phe Val Thr
Thr Ser Gln Gln Lys 90 95 100 aac atc ggc tcg cgg ttg tat atg atg
aaa gac gat tcc act tac gag 432Asn Ile Gly Ser Arg Leu Tyr Met Met
Lys Asp Asp Ser Thr Tyr Glu 105 110 115 atg ttc aag ctc ctc aac cag
gaa ttc act ttc gat gtc gac gtc tcc 480Met Phe Lys Leu Leu Asn Gln
Glu Phe Thr Phe Asp Val Asp Val Ser 120 125 130 aac ctc cct tgt ggc
ttg aac gga gcg ctc tac ttc gtc gcc atg gat 528Asn Leu Pro Cys Gly
Leu Asn Gly Ala Leu Tyr Phe Val Ala Met Asp 135 140 145 150 gcg gat
gga ggc atg tcc aag tat cct acc aac aaa gca gga gcc aag 576Ala Asp
Gly Gly Met Ser Lys Tyr Pro Thr Asn Lys Ala Gly Ala Lys 155 160 165
tat ggt aca ggt tac tgt gat tcc cag tgt ccc agg gat ctc aag ttc
624Tyr Gly Thr Gly Tyr Cys Asp Ser Gln Cys Pro Arg Asp Leu Lys Phe
170 175 180 atc aac ggt cag gcc aac gtc gag ggt tgg cag cct tcg tcg
aac gat 672Ile Asn Gly Gln Ala Asn Val Glu Gly Trp Gln Pro Ser Ser
Asn Asp 185 190 195 gcc aac gca ggt acc ggc aac cac ggt tcc tgt tgt
gcc gaa atg gac 720Ala Asn Ala Gly Thr Gly Asn His Gly Ser Cys Cys
Ala Glu Met Asp 200 205 210 att tgg gaa gcg aac tcg atc tcg acg gcg
ttc act cct cac ccg tgt 768Ile Trp Glu Ala Asn Ser Ile Ser Thr Ala
Phe Thr Pro His Pro Cys 215 220 225 230 gat aca ccc gga cag gtg atg
tgt aca ggc gac gcc tgt ggc gga acc 816Asp Thr Pro Gly Gln Val Met
Cys Thr Gly Asp Ala Cys Gly Gly Thr 235 240 245 tac tcg tcg gat cga
tat ggc ggt acg tgt gac ccc gac ggc tgt gac 864Tyr Ser Ser Asp Arg
Tyr Gly Gly Thr Cys Asp Pro Asp Gly Cys Asp 250 255 260 ttc aac tcc
ttc agg cag ggc aac aaa aca ttc tat gga cct ggc atg 912Phe Asn Ser
Phe Arg Gln Gly Asn Lys Thr Phe Tyr Gly Pro Gly Met 265 270 275 acg
gtg gat aca aag tcg aaa ttc aca gtc gtc act cag ttc atc acc 960Thr
Val Asp Thr Lys Ser Lys Phe Thr Val Val Thr Gln Phe Ile Thr 280 285
290 gac gat ggt acg tcc tcg ggt acc ttg aag gag atc aaa agg ttc tat
1008Asp Asp Gly Thr Ser Ser Gly Thr Leu Lys Glu Ile Lys Arg Phe Tyr
295 300 305 310 gtc cag aac gga aag gtc atc ccg aac tcg gag tcc acg
tgg aca gga 1056Val Gln Asn Gly Lys Val Ile Pro Asn Ser Glu Ser Thr
Trp Thr Gly 315 320 325 gtg tcg ggt aac tcc atc act acg gag tat tgt
aca gcc cag aag tcg 1104Val Ser Gly Asn Ser Ile Thr Thr Glu Tyr Cys
Thr Ala Gln Lys Ser 330 335 340 ctc ttc cag gat cag aac gtc ttc gag
aaa cat gga ggc ttg gaa gga 1152Leu Phe Gln Asp Gln Asn Val Phe Glu
Lys His Gly Gly Leu Glu Gly 345 350 355 atg ggt gcc gca ttg gcc cag
ggt atg gtc ctc gtc atg tcc ttg tgg 1200Met Gly Ala Ala Leu Ala Gln
Gly Met Val Leu Val Met Ser Leu Trp 360 365 370 gac gac cac tcg gcc
aac atg ctc tgg ttg gat tcc aac tac ccc acc 1248Asp Asp His Ser Ala
Asn Met Leu Trp Leu Asp Ser Asn Tyr Pro Thr 375 380 385 390 act gcc
tcg tcc acg aca ccg ggt gtc gca cgc gga act tgt gat atc 1296Thr Ala
Ser Ser Thr Thr Pro Gly Val Ala Arg Gly Thr Cys Asp Ile 395 400 405
tcc tcg gga gtg cct gca gac gtc gag gcg aac cat ccc gac gcc tac
1344Ser Ser Gly Val Pro Ala Asp Val Glu Ala Asn His Pro Asp Ala Tyr
410 415 420 gtg gtc tac tcg aac att aag gtg gga ccc atc ggt tcg aca
ttc aac 1392Val Val Tyr Ser Asn Ile Lys Val Gly Pro Ile Gly Ser Thr
Phe Asn 425 430 435 tcc gga ggc tcg aac cct gga ggc gga acg acc act
act aca acg act 1440Ser Gly Gly Ser Asn Pro Gly Gly Gly Thr Thr Thr
Thr Thr Thr Thr 440 445 450 cag ccg aca aca aca act acc aca gca ggc
aac cct gga ggt aca ggt 1488Gln Pro Thr Thr Thr Thr Thr Thr Ala Gly
Asn Pro Gly Gly Thr Gly 455 460 465 470 gtg gcc cag cac tac gga cag
tgt ggc ggt atc gga tgg aca gga cct 1536Val Ala Gln His Tyr Gly Gln
Cys Gly Gly Ile Gly Trp Thr Gly Pro 475 480 485 act act tgt gca tcg
cct tat acc tgt cag aaa ttg aac gac tac tac 1584Thr Thr Cys Ala Ser
Pro Tyr Thr Cys Gln Lys Leu Asn Asp Tyr Tyr 490 495 500 tcg cag tgt
ttg taa 1599Ser Gln Cys Leu 505 26532PRTAspergillus fumigatus 26Met
Leu Ala Ser Thr Phe Ser Tyr Arg Met Tyr Lys Thr Ala Leu Ile -25 -20
-15 Leu Ala Ala Leu Leu Gly Ser Gly Gln Ala Gln Gln Val Gly Thr Ser
-10 -5 -1 1 5 Gln Ala Glu Val His Pro Ser Met Thr Trp Gln Ser Cys
Thr Ala Gly 10 15 20 Gly Ser Cys Thr Thr Asn Asn Gly Lys Val Val
Ile Asp Ala Asn Trp 25 30 35 Arg Trp Val His Lys Thr Gly Asp Tyr
Thr Asn Cys Tyr Thr Gly Asn 40 45 50 Thr Trp Asp Thr Thr Ile Cys
Pro Asp Asp Ala Thr Cys Ala Ser Asn 55 60 65 70 Cys Ala Leu Glu Gly
Ala Asn Tyr Glu Ser Thr Tyr Gly Val Thr Ala 75 80 85 Ser Gly Asn
Ser Leu Arg Leu Asn Phe Val Thr Thr Ser Gln Gln Lys 90 95 100 Asn
Ile Gly Ser Arg Leu Tyr Met Met Lys Asp Asp Ser Thr Tyr Glu 105 110
115 Met Phe Lys Leu Leu Asn Gln Glu Phe Thr Phe Asp Val Asp Val Ser
120 125 130 Asn Leu Pro Cys Gly Leu Asn Gly Ala Leu Tyr Phe Val Ala
Met Asp 135 140 145 150 Ala Asp Gly Gly Met Ser Lys Tyr Pro Thr Asn
Lys Ala Gly Ala Lys 155 160 165 Tyr Gly Thr Gly Tyr Cys Asp Ser Gln
Cys Pro Arg Asp Leu Lys Phe 170 175 180 Ile Asn Gly Gln Ala Asn Val
Glu Gly Trp Gln Pro Ser Ser Asn Asp 185 190 195 Ala Asn Ala Gly Thr
Gly Asn His Gly Ser Cys Cys Ala Glu Met Asp 200 205 210 Ile Trp Glu
Ala Asn Ser Ile Ser Thr Ala Phe Thr Pro His Pro Cys 215 220 225 230
Asp Thr Pro Gly Gln Val Met Cys Thr Gly Asp Ala Cys Gly Gly Thr 235
240 245 Tyr Ser Ser Asp Arg Tyr Gly Gly Thr Cys Asp Pro Asp Gly Cys
Asp 250 255 260 Phe Asn Ser Phe Arg Gln Gly Asn Lys Thr Phe Tyr Gly
Pro Gly Met 265 270 275 Thr Val Asp Thr Lys Ser Lys Phe Thr Val Val
Thr Gln Phe Ile Thr 280 285 290 Asp Asp Gly Thr Ser Ser Gly Thr Leu
Lys Glu Ile Lys Arg Phe Tyr 295 300 305 310 Val Gln Asn Gly Lys Val
Ile Pro Asn Ser Glu Ser Thr Trp Thr Gly 315 320 325 Val Ser Gly Asn
Ser Ile Thr Thr Glu Tyr Cys Thr Ala Gln Lys Ser 330 335 340 Leu Phe
Gln Asp Gln Asn Val Phe Glu Lys His Gly Gly Leu Glu Gly 345 350 355
Met Gly Ala Ala Leu Ala Gln Gly Met Val Leu Val Met Ser Leu Trp 360
365 370 Asp Asp His Ser Ala Asn Met Leu Trp Leu Asp Ser Asn Tyr Pro
Thr 375 380 385 390 Thr Ala Ser Ser Thr Thr Pro Gly Val Ala Arg Gly
Thr Cys Asp Ile 395 400 405 Ser Ser Gly Val Pro Ala Asp Val Glu Ala
Asn His Pro Asp Ala Tyr 410 415 420 Val Val Tyr Ser Asn Ile Lys Val
Gly Pro Ile Gly Ser Thr Phe Asn 425 430 435 Ser Gly Gly Ser Asn Pro
Gly Gly Gly Thr Thr Thr Thr Thr Thr Thr 440 445 450 Gln Pro Thr Thr
Thr Thr Thr Thr Ala Gly Asn Pro Gly Gly Thr Gly 455 460 465 470 Val
Ala Gln His Tyr Gly Gln Cys Gly Gly Ile Gly Trp Thr Gly Pro 475 480
485 Thr Thr Cys Ala Ser Pro Tyr Thr Cys Gln Lys Leu Asn Asp Tyr Tyr
490 495 500 Ser Gln Cys Leu 505 271599DNAAspergillus
fumigatussig_peptide(1)..(26)CDS(1)..(1596)mat_peptide(79)..(1596)
27atg ttg gcc tcc acg ttc tcc tat cgc atg tac aaa aca gcg ctc atc
48Met Leu Ala Ser Thr Phe Ser Tyr Arg Met Tyr Lys Thr Ala Leu Ile
-25 -20 -15 ttg gca gcc ctc ttg ggc tcg gga cag gca cag cag gtc gga
acc tcg 96Leu Ala Ala Leu Leu Gly Ser Gly Gln Ala Gln Gln Val Gly
Thr Ser -10 -5 -1 1 5 cag gcc gag gtc cat cct tcc atg acg tgg cag
tcg tgt aca gcg ggt 144Gln Ala Glu Val His Pro Ser Met Thr Trp Gln
Ser Cys Thr Ala Gly 10 15 20 ggt tcg tgt acc aca aac aac ggt aaa
gtc gtg atc gat gca aac tgg 192Gly Ser Cys Thr Thr Asn Asn Gly Lys
Val Val Ile Asp Ala Asn Trp 25 30 35 agg tgg gtg cac aag gtc ggc
gac tac acc aac tgt tac aca ggc aac 240Arg Trp Val His Lys Val Gly
Asp Tyr Thr Asn Cys Tyr Thr Gly Asn 40 45 50 aca tgg gat aca acc
atc tgt ccc gac gat gcc act tgt gca tcc aac 288Thr Trp Asp Thr Thr
Ile Cys Pro Asp Asp Ala Thr Cys Ala Ser Asn 55 60 65 70 tgt gca ctc
gag ggt gcc aac tat gag tcg acg tac gga gtg acc gcc 336Cys Ala Leu
Glu Gly Ala Asn Tyr Glu Ser Thr Tyr Gly Val Thr Ala 75 80 85 tcc
gga aac tcg ctc agg ctc aac ttc gtc aca act tcc cag cag aag 384Ser
Gly Asn Ser Leu Arg Leu Asn Phe Val Thr Thr Ser Gln Gln Lys 90 95
100 aac atc ggc tcg cgg ttg tat atg atg aaa gac gat tcc act tac gag
432Asn Ile Gly Ser Arg Leu Tyr Met Met Lys Asp Asp Ser Thr Tyr Glu
105 110 115 atg ttc aag ctc ctc aac cag gaa ttc act ttc gat gtc gac
gtc tcc 480Met Phe Lys Leu Leu Asn Gln Glu Phe Thr Phe Asp Val Asp
Val Ser 120 125 130 aac ctc cct tgt ggc ttg aac gga gcg ctc tac ttc
gtc gcc atg gat 528Asn Leu Pro Cys Gly Leu Asn Gly Ala Leu Tyr Phe
Val Ala Met Asp 135 140 145 150 gcg gat gga ggc atg tcc aag tat cct
acc aac aaa gca gga gcc aag 576Ala Asp Gly Gly Met Ser Lys Tyr Pro
Thr Asn Lys Ala Gly Ala Lys 155 160 165 tat ggt aca ggt tac tgt gat
tcc cag tgt ccc agg gat ctc aag ttc 624Tyr Gly Thr Gly Tyr Cys Asp
Ser Gln Cys Pro Arg Asp Leu Lys Phe 170 175 180 atc aac ggt cag gcc
aac gtc gag ggt tgg cag cct tcg tcg aac gat 672Ile Asn Gly Gln Ala
Asn Val Glu Gly Trp Gln Pro Ser Ser Asn Asp 185 190 195 gcc aac gca
ggt acc ggc aac cac ggt tcc tgt tgt gcc gaa atg gac 720Ala Asn Ala
Gly Thr Gly Asn His Gly Ser Cys Cys Ala Glu Met Asp 200 205 210 att
tgg gaa gcg aac tcg atc tcg acg gcg ttc act cct cac ccg tgt 768Ile
Trp Glu Ala Asn Ser Ile Ser Thr Ala Phe Thr Pro His Pro Cys 215 220
225 230 gat aca ccc gga cag gtg atg tgt aca ggc gac gcc tgt ggc gga
acc 816Asp Thr Pro Gly Gln Val Met Cys Thr Gly Asp Ala Cys Gly Gly
Thr 235 240 245 tac tcg tcg gat cga tat ggc ggt acg tgt gac ccc gac
ggc tgt gac 864Tyr Ser Ser Asp Arg Tyr Gly Gly Thr Cys Asp Pro Asp
Gly Cys Asp 250 255 260 ttc aac cct ttc agg cag ggc aac aaa aca ttc
tat gga cct ggc atg 912Phe Asn Pro Phe Arg Gln Gly Asn Lys Thr Phe
Tyr Gly Pro Gly Met 265 270 275 acg gtg gat aca aag tcg aaa ttc aca
gtc gtc act cag ttc atc acc 960Thr Val Asp Thr Lys Ser Lys Phe Thr
Val Val Thr Gln Phe Ile Thr 280 285 290 gac gat ggt acg tcc tcg ggt
acc ttg aag gag atc aaa agg ttc tat 1008Asp Asp Gly Thr Ser Ser Gly
Thr Leu Lys Glu Ile Lys Arg Phe Tyr 295 300 305 310 gtc cag aac gga
aag gtc atc ccg aac tcg gag tcc acg tgg aca gga 1056Val Gln Asn Gly
Lys Val Ile Pro Asn Ser Glu Ser Thr Trp Thr Gly 315 320 325 gtg tcg
ggt aac tcc atc act acg gag tat tgt aca gcc cag aag tcg 1104Val Ser
Gly Asn Ser Ile Thr Thr Glu Tyr Cys Thr Ala Gln Lys Ser 330 335 340
ctc ttc cag gat cag aac gtc ttc gag aaa cat gga ggc ttg gaa gga
1152Leu Phe Gln Asp Gln Asn Val Phe Glu Lys His Gly Gly Leu Glu Gly
345 350 355 atg ggt gcc gca ttg gcc cag ggt atg gtc ctc gtc atg tcc
ttg tgg 1200Met Gly Ala Ala Leu Ala Gln Gly Met Val Leu Val Met Ser
Leu Trp 360 365 370 gac gac cac tcg gcc aac atg ctc tgg ttg gat tcc
aac tac ccc acc 1248Asp Asp His Ser Ala Asn Met Leu Trp Leu Asp Ser
Asn Tyr Pro Thr 375 380 385 390 act gcc tcg tcc acg aca ccg ggt gtc
gca cgc gga act tgt gat atc 1296Thr Ala Ser Ser Thr Thr Pro Gly Val
Ala Arg Gly Thr Cys Asp Ile 395 400 405 tcc tcg gga gtg cct gca gac
gtc gag gcg aac cat ccc gac gcc tac 1344Ser Ser Gly Val Pro Ala Asp
Val Glu Ala Asn His Pro Asp Ala Tyr 410 415 420 gtg gtc tac tcg aac
att aag gtg gga ccc atc ggt tcg aca ttc aac 1392Val Val Tyr Ser Asn
Ile Lys Val Gly Pro Ile Gly Ser Thr Phe Asn 425 430 435 tcc gga ggc
tcg aac cct gga ggc gga acg acc act act aca acg act 1440Ser Gly Gly
Ser Asn Pro Gly Gly Gly Thr Thr Thr Thr Thr Thr Thr 440 445 450 cag
ccg aca aca aca act acc aca gca ggc aac cct gga ggt aca ggt 1488Gln
Pro Thr Thr Thr Thr Thr Thr Ala Gly Asn Pro Gly Gly Thr Gly 455 460
465 470 gtg gcc cag cac tac gga cag tgt ggc ggt atc gga tgg aca gga
cct 1536Val Ala Gln His Tyr Gly Gln Cys Gly Gly Ile Gly Trp Thr Gly
Pro 475 480 485 act act tgt gca tcg cct tat acc tgt cag aaa ttg aac
gac tac tac 1584Thr Thr Cys Ala Ser Pro Tyr Thr Cys Gln Lys Leu Asn
Asp Tyr Tyr 490 495 500 tcg cag tgt ttg taa 1599Ser Gln Cys Leu 505
28532PRTAspergillus fumigatus 28Met Leu Ala Ser Thr Phe Ser Tyr Arg
Met Tyr Lys Thr Ala Leu Ile -25 -20 -15 Leu Ala Ala Leu Leu Gly Ser
Gly Gln Ala Gln Gln Val Gly Thr
Ser -10 -5 -1 1 5 Gln Ala Glu Val His Pro Ser Met Thr Trp Gln Ser
Cys Thr Ala Gly 10 15 20 Gly Ser Cys Thr Thr Asn Asn Gly Lys Val
Val Ile Asp Ala Asn Trp 25 30 35 Arg Trp Val His Lys Val Gly Asp
Tyr Thr Asn Cys Tyr Thr Gly Asn 40 45 50 Thr Trp Asp Thr Thr Ile
Cys Pro Asp Asp Ala Thr Cys Ala Ser Asn 55 60 65 70 Cys Ala Leu Glu
Gly Ala Asn Tyr Glu Ser Thr Tyr Gly Val Thr Ala 75 80 85 Ser Gly
Asn Ser Leu Arg Leu Asn Phe Val Thr Thr Ser Gln Gln Lys 90 95 100
Asn Ile Gly Ser Arg Leu Tyr Met Met Lys Asp Asp Ser Thr Tyr Glu 105
110 115 Met Phe Lys Leu Leu Asn Gln Glu Phe Thr Phe Asp Val Asp Val
Ser 120 125 130 Asn Leu Pro Cys Gly Leu Asn Gly Ala Leu Tyr Phe Val
Ala Met Asp 135 140 145 150 Ala Asp Gly Gly Met Ser Lys Tyr Pro Thr
Asn Lys Ala Gly Ala Lys 155 160 165 Tyr Gly Thr Gly Tyr Cys Asp Ser
Gln Cys Pro Arg Asp Leu Lys Phe 170 175 180 Ile Asn Gly Gln Ala Asn
Val Glu Gly Trp Gln Pro Ser Ser Asn Asp 185 190 195 Ala Asn Ala Gly
Thr Gly Asn His Gly Ser Cys Cys Ala Glu Met Asp 200 205 210 Ile Trp
Glu Ala Asn Ser Ile Ser Thr Ala Phe Thr Pro His Pro Cys 215 220 225
230 Asp Thr Pro Gly Gln Val Met Cys Thr Gly Asp Ala Cys Gly Gly Thr
235 240 245 Tyr Ser Ser Asp Arg Tyr Gly Gly Thr Cys Asp Pro Asp Gly
Cys Asp 250 255 260 Phe Asn Pro Phe Arg Gln Gly Asn Lys Thr Phe Tyr
Gly Pro Gly Met 265 270 275 Thr Val Asp Thr Lys Ser Lys Phe Thr Val
Val Thr Gln Phe Ile Thr 280 285 290 Asp Asp Gly Thr Ser Ser Gly Thr
Leu Lys Glu Ile Lys Arg Phe Tyr 295 300 305 310 Val Gln Asn Gly Lys
Val Ile Pro Asn Ser Glu Ser Thr Trp Thr Gly 315 320 325 Val Ser Gly
Asn Ser Ile Thr Thr Glu Tyr Cys Thr Ala Gln Lys Ser 330 335 340 Leu
Phe Gln Asp Gln Asn Val Phe Glu Lys His Gly Gly Leu Glu Gly 345 350
355 Met Gly Ala Ala Leu Ala Gln Gly Met Val Leu Val Met Ser Leu Trp
360 365 370 Asp Asp His Ser Ala Asn Met Leu Trp Leu Asp Ser Asn Tyr
Pro Thr 375 380 385 390 Thr Ala Ser Ser Thr Thr Pro Gly Val Ala Arg
Gly Thr Cys Asp Ile 395 400 405 Ser Ser Gly Val Pro Ala Asp Val Glu
Ala Asn His Pro Asp Ala Tyr 410 415 420 Val Val Tyr Ser Asn Ile Lys
Val Gly Pro Ile Gly Ser Thr Phe Asn 425 430 435 Ser Gly Gly Ser Asn
Pro Gly Gly Gly Thr Thr Thr Thr Thr Thr Thr 440 445 450 Gln Pro Thr
Thr Thr Thr Thr Thr Ala Gly Asn Pro Gly Gly Thr Gly 455 460 465 470
Val Ala Gln His Tyr Gly Gln Cys Gly Gly Ile Gly Trp Thr Gly Pro 475
480 485 Thr Thr Cys Ala Ser Pro Tyr Thr Cys Gln Lys Leu Asn Asp Tyr
Tyr 490 495 500 Ser Gln Cys Leu 505 2921DNAAspergillus oryzae
29cccttgtcga tgcgatgtat c 213021DNAAspergillus oryzae 30atcctcaatt
ccgtcggtcg a 213125DNAAspergillus oryzae 31gtgatacacc cggacaggtg
atgtg 253225DNAAspergillus oryzae 32ccatatcgat ccgacgagta ggttc
253346DNAAspergillus fumigatus 33aactggaggt gggtgcacaa gacaggcgac
tacaccaact gttaca 463433DNAAspergillus fumigatus 34cttgtgcacc
cacctccagt ttgcatcgat cac 333547DNAAspergillus fumigatus
35cccgacggct gtgacttcaa ccctttcagg cagggcaaca aaacatt
473632DNAAspergillus fumigatus 36gttgaagtca cagccgtcgg ggtcacacgt
ac 32371599DNAAspergillus
fumigatussig_peptide(1)..(26)CDS(1)..(1596)mat_peptide(79)..(1596)
37atg ttg gcc tcc acg ttc tcc tat cgc atg tac aaa aca gcg ctc atc
48Met Leu Ala Ser Thr Phe Ser Tyr Arg Met Tyr Lys Thr Ala Leu Ile
-25 -20 -15 ttg gca gcc ctc ttg ggc tcg gga cag gca cag cag gtc gga
acc tcg 96Leu Ala Ala Leu Leu Gly Ser Gly Gln Ala Gln Gln Val Gly
Thr Ser -10 -5 -1 1 5 cag gcc gag gtc cat cct tcc atg acg tgg cag
tcg tgt aca gcg ggt 144Gln Ala Glu Val His Pro Ser Met Thr Trp Gln
Ser Cys Thr Ala Gly 10 15 20 ggt tcg tgt acc aca aac aac ggt aaa
gtc gtg atc gat gca aac tgg 192Gly Ser Cys Thr Thr Asn Asn Gly Lys
Val Val Ile Asp Ala Asn Trp 25 30 35 agg tgg gtg cac aag gtc ggc
gac tac acc aac tgt tac aca ggc aac 240Arg Trp Val His Lys Val Gly
Asp Tyr Thr Asn Cys Tyr Thr Gly Asn 40 45 50 aca tgg gat aca acc
atc tgt ccc gac gat gcc act tgt gca tcc aac 288Thr Trp Asp Thr Thr
Ile Cys Pro Asp Asp Ala Thr Cys Ala Ser Asn 55 60 65 70 tgt gca ctc
gag ggt gcc aac tat gag tcg acg tac gga gtg acc gcc 336Cys Ala Leu
Glu Gly Ala Asn Tyr Glu Ser Thr Tyr Gly Val Thr Ala 75 80 85 tcc
gga aac tcg ctc agg ctc aac ttc gtc aca act tcc cag cag aag 384Ser
Gly Asn Ser Leu Arg Leu Asn Phe Val Thr Thr Ser Gln Gln Lys 90 95
100 aac atc ggc tcg cgg ttg tat atg atg aaa gac gat tcc act tac gag
432Asn Ile Gly Ser Arg Leu Tyr Met Met Lys Asp Asp Ser Thr Tyr Glu
105 110 115 atg ttc aag ctc ctc aac cag gaa ttc act ttc gat gtc gac
gtc tcc 480Met Phe Lys Leu Leu Asn Gln Glu Phe Thr Phe Asp Val Asp
Val Ser 120 125 130 aac ctc cct tgt ggc ttg aac gga gcg ctc tac ttc
gtc gcc atg gat 528Asn Leu Pro Cys Gly Leu Asn Gly Ala Leu Tyr Phe
Val Ala Met Asp 135 140 145 150 gcg gat gga ggc atg tcc aag tat cct
acc aac aaa gca gga gcc aag 576Ala Asp Gly Gly Met Ser Lys Tyr Pro
Thr Asn Lys Ala Gly Ala Lys 155 160 165 tat ggt aca ggt tac tgt gat
tcc cag tgt ccc agg gat ctc aag ttc 624Tyr Gly Thr Gly Tyr Cys Asp
Ser Gln Cys Pro Arg Asp Leu Lys Phe 170 175 180 atc aac ggt cag gcc
aac gtc gag ggt tgg cag cct tcg tcg aac gat 672Ile Asn Gly Gln Ala
Asn Val Glu Gly Trp Gln Pro Ser Ser Asn Asp 185 190 195 gcc aac gca
ggt acc ggc aac cac ggt tcc tgt tgt gcc gaa atg gac 720Ala Asn Ala
Gly Thr Gly Asn His Gly Ser Cys Cys Ala Glu Met Asp 200 205 210 att
tgg gaa gcg aac tcg atc tcg acg gcg ttc act cct cac ccg tgt 768Ile
Trp Glu Ala Asn Ser Ile Ser Thr Ala Phe Thr Pro His Pro Cys 215 220
225 230 gat aca ccc gga cag gtg atg tgt aca ggc gac gcc tgt ggc gga
acc 816Asp Thr Pro Gly Gln Val Met Cys Thr Gly Asp Ala Cys Gly Gly
Thr 235 240 245 tac tcg tcg gat cga tat ggc ggt acg tgt gac ccc gac
ggc tgt gac 864Tyr Ser Ser Asp Arg Tyr Gly Gly Thr Cys Asp Pro Asp
Gly Cys Asp 250 255 260 ttc aac tcc ttc agg cag ggc aac aaa aca ttc
tat gga cct ggc atg 912Phe Asn Ser Phe Arg Gln Gly Asn Lys Thr Phe
Tyr Gly Pro Gly Met 265 270 275 acg gtg gat aca aag tcg aaa ttc aca
gtc gtc act cag ttc atc acc 960Thr Val Asp Thr Lys Ser Lys Phe Thr
Val Val Thr Gln Phe Ile Thr 280 285 290 gac gat ggt acg tcc tcg ggt
acc ttg aag gag atc aaa agg ttc tat 1008Asp Asp Gly Thr Ser Ser Gly
Thr Leu Lys Glu Ile Lys Arg Phe Tyr 295 300 305 310 gtc cag aac gga
aag gtc atc ccg aac tcg gag tcc acg tgg aca gga 1056Val Gln Asn Gly
Lys Val Ile Pro Asn Ser Glu Ser Thr Trp Thr Gly 315 320 325 gtg tcg
ggt aac tcc atc act acg gag tat tgt aca gcc cag aag tcg 1104Val Ser
Gly Asn Ser Ile Thr Thr Glu Tyr Cys Thr Ala Gln Lys Ser 330 335 340
ctc ttc cag gat cag aac gtc ttc gag aaa cat gga ggc ttg gaa gga
1152Leu Phe Gln Asp Gln Asn Val Phe Glu Lys His Gly Gly Leu Glu Gly
345 350 355 atg ggt gcc gca ttg gcc cag ggt atg gtc ctc gtc atg tcc
ttg tgg 1200Met Gly Ala Ala Leu Ala Gln Gly Met Val Leu Val Met Ser
Leu Trp 360 365 370 gac gac cac tcg gcc aac atg ctc tgg ttg gat tcc
aac tac ccc acc 1248Asp Asp His Ser Ala Asn Met Leu Trp Leu Asp Ser
Asn Tyr Pro Thr 375 380 385 390 gat gcc gat cct acg aca ccg ggt gtc
gca cgc gga act tgt gat atc 1296Asp Ala Asp Pro Thr Thr Pro Gly Val
Ala Arg Gly Thr Cys Asp Ile 395 400 405 tcc tcg gga gtg cct gca gac
gtc gag gcg aac cat ccc gac gcc tac 1344Ser Ser Gly Val Pro Ala Asp
Val Glu Ala Asn His Pro Asp Ala Tyr 410 415 420 gtg gtc tac tcg aac
att aag gtg gga ccc atc ggt tcg aca ttc aac 1392Val Val Tyr Ser Asn
Ile Lys Val Gly Pro Ile Gly Ser Thr Phe Asn 425 430 435 tcc gga ggc
tcg aac cct gga ggc gga acg acc act act aca acg act 1440Ser Gly Gly
Ser Asn Pro Gly Gly Gly Thr Thr Thr Thr Thr Thr Thr 440 445 450 cag
ccg aca aca aca act acc aca gca ggc aac cct gga ggt aca ggt 1488Gln
Pro Thr Thr Thr Thr Thr Thr Ala Gly Asn Pro Gly Gly Thr Gly 455 460
465 470 gtg gcc cag cac tac gga cag tgt ggc ggt atc gga tgg aca gga
cct 1536Val Ala Gln His Tyr Gly Gln Cys Gly Gly Ile Gly Trp Thr Gly
Pro 475 480 485 act act tgt gca tcg cct tat acc tgt cag aaa ttg aac
gac tac tac 1584Thr Thr Cys Ala Ser Pro Tyr Thr Cys Gln Lys Leu Asn
Asp Tyr Tyr 490 495 500 tcg cag tgt ttg taa 1599Ser Gln Cys Leu 505
38532PRTAspergillus fumigatus 38Met Leu Ala Ser Thr Phe Ser Tyr Arg
Met Tyr Lys Thr Ala Leu Ile -25 -20 -15 Leu Ala Ala Leu Leu Gly Ser
Gly Gln Ala Gln Gln Val Gly Thr Ser -10 -5 -1 1 5 Gln Ala Glu Val
His Pro Ser Met Thr Trp Gln Ser Cys Thr Ala Gly 10 15 20 Gly Ser
Cys Thr Thr Asn Asn Gly Lys Val Val Ile Asp Ala Asn Trp 25 30 35
Arg Trp Val His Lys Val Gly Asp Tyr Thr Asn Cys Tyr Thr Gly Asn 40
45 50 Thr Trp Asp Thr Thr Ile Cys Pro Asp Asp Ala Thr Cys Ala Ser
Asn 55 60 65 70 Cys Ala Leu Glu Gly Ala Asn Tyr Glu Ser Thr Tyr Gly
Val Thr Ala 75 80 85 Ser Gly Asn Ser Leu Arg Leu Asn Phe Val Thr
Thr Ser Gln Gln Lys 90 95 100 Asn Ile Gly Ser Arg Leu Tyr Met Met
Lys Asp Asp Ser Thr Tyr Glu 105 110 115 Met Phe Lys Leu Leu Asn Gln
Glu Phe Thr Phe Asp Val Asp Val Ser 120 125 130 Asn Leu Pro Cys Gly
Leu Asn Gly Ala Leu Tyr Phe Val Ala Met Asp 135 140 145 150 Ala Asp
Gly Gly Met Ser Lys Tyr Pro Thr Asn Lys Ala Gly Ala Lys 155 160 165
Tyr Gly Thr Gly Tyr Cys Asp Ser Gln Cys Pro Arg Asp Leu Lys Phe 170
175 180 Ile Asn Gly Gln Ala Asn Val Glu Gly Trp Gln Pro Ser Ser Asn
Asp 185 190 195 Ala Asn Ala Gly Thr Gly Asn His Gly Ser Cys Cys Ala
Glu Met Asp 200 205 210 Ile Trp Glu Ala Asn Ser Ile Ser Thr Ala Phe
Thr Pro His Pro Cys 215 220 225 230 Asp Thr Pro Gly Gln Val Met Cys
Thr Gly Asp Ala Cys Gly Gly Thr 235 240 245 Tyr Ser Ser Asp Arg Tyr
Gly Gly Thr Cys Asp Pro Asp Gly Cys Asp 250 255 260 Phe Asn Ser Phe
Arg Gln Gly Asn Lys Thr Phe Tyr Gly Pro Gly Met 265 270 275 Thr Val
Asp Thr Lys Ser Lys Phe Thr Val Val Thr Gln Phe Ile Thr 280 285 290
Asp Asp Gly Thr Ser Ser Gly Thr Leu Lys Glu Ile Lys Arg Phe Tyr 295
300 305 310 Val Gln Asn Gly Lys Val Ile Pro Asn Ser Glu Ser Thr Trp
Thr Gly 315 320 325 Val Ser Gly Asn Ser Ile Thr Thr Glu Tyr Cys Thr
Ala Gln Lys Ser 330 335 340 Leu Phe Gln Asp Gln Asn Val Phe Glu Lys
His Gly Gly Leu Glu Gly 345 350 355 Met Gly Ala Ala Leu Ala Gln Gly
Met Val Leu Val Met Ser Leu Trp 360 365 370 Asp Asp His Ser Ala Asn
Met Leu Trp Leu Asp Ser Asn Tyr Pro Thr 375 380 385 390 Asp Ala Asp
Pro Thr Thr Pro Gly Val Ala Arg Gly Thr Cys Asp Ile 395 400 405 Ser
Ser Gly Val Pro Ala Asp Val Glu Ala Asn His Pro Asp Ala Tyr 410 415
420 Val Val Tyr Ser Asn Ile Lys Val Gly Pro Ile Gly Ser Thr Phe Asn
425 430 435 Ser Gly Gly Ser Asn Pro Gly Gly Gly Thr Thr Thr Thr Thr
Thr Thr 440 445 450 Gln Pro Thr Thr Thr Thr Thr Thr Ala Gly Asn Pro
Gly Gly Thr Gly 455 460 465 470 Val Ala Gln His Tyr Gly Gln Cys Gly
Gly Ile Gly Trp Thr Gly Pro 475 480 485 Thr Thr Cys Ala Ser Pro Tyr
Thr Cys Gln Lys Leu Asn Asp Tyr Tyr 490 495 500 Ser Gln Cys Leu 505
3954DNAAspergillus fumigatus 39gttggattcc aactacccca ccgatgccga
tcctacgaca ccgggtgtcg cacg 544034DNAAspergillus fumigatus
40ggtggggtag ttggaatcca accagagcat gttg 34411599DNAAspergillus
fumigatussig_peptide(1)..(26)CDS(1)..(1596)mat_peptide(79)..(1596)
41atg ttg gcc tcc acg ttc tcc tat cgc atg tac aaa aca gcg ctc atc
48Met Leu Ala Ser Thr Phe Ser Tyr Arg Met Tyr Lys Thr Ala Leu Ile
-25 -20 -15 ttg gca gcc ctc ttg ggc tcg gga cag gca cag cag gtc gga
acc tcg 96Leu Ala Ala Leu Leu Gly Ser Gly Gln Ala Gln Gln Val Gly
Thr Ser -10 -5 -1 1 5 cag gcc gag gtc cat cct tcc atg acg tgg cag
tcg tgt aca gcg ggt 144Gln Ala Glu Val His Pro Ser Met Thr Trp Gln
Ser Cys Thr Ala Gly 10 15 20 ggt tcg tgt acc aca aac aac ggt aaa
gtc gtg atc gat gca aac tgg 192Gly Ser Cys Thr Thr Asn Asn Gly Lys
Val Val Ile Asp Ala Asn Trp 25 30 35 agg tgg gtg cac aag aca ggc
gac tac acc aac tgt tac aca ggc aac 240Arg Trp Val His Lys Thr Gly
Asp Tyr Thr Asn Cys Tyr Thr Gly Asn 40 45 50 aca tgg gat aca acc
atc tgt ccc gac gat gcc act tgt gca tcc aac 288Thr Trp Asp Thr Thr
Ile Cys Pro Asp Asp Ala Thr Cys Ala Ser Asn 55 60 65 70 tgt gca ctc
gag ggt
gcc aac tat gag tcg acg tac gga gtg acc gcc 336Cys Ala Leu Glu Gly
Ala Asn Tyr Glu Ser Thr Tyr Gly Val Thr Ala 75 80 85 tcc gga aac
tcg ctc agg ctc aac ttc gtc aca act tcc cag cag aag 384Ser Gly Asn
Ser Leu Arg Leu Asn Phe Val Thr Thr Ser Gln Gln Lys 90 95 100 aac
atc ggc tcg cgg ttg tat atg atg aaa gac gat tcc act tac gag 432Asn
Ile Gly Ser Arg Leu Tyr Met Met Lys Asp Asp Ser Thr Tyr Glu 105 110
115 atg ttc aag ctc ctc aac cag gaa ttc act ttc gat gtc gac gtc tcc
480Met Phe Lys Leu Leu Asn Gln Glu Phe Thr Phe Asp Val Asp Val Ser
120 125 130 aac ctc cct tgt ggc ttg aac gga gcg ctc tac ttc gtc gcc
atg gat 528Asn Leu Pro Cys Gly Leu Asn Gly Ala Leu Tyr Phe Val Ala
Met Asp 135 140 145 150 gcg gat gga ggc atg tcc aag tat cct acc aac
aaa gca gga gcc aag 576Ala Asp Gly Gly Met Ser Lys Tyr Pro Thr Asn
Lys Ala Gly Ala Lys 155 160 165 tat ggt aca ggt tac tgt gat tcc cag
tgt ccc agg gat ctc aag ttc 624Tyr Gly Thr Gly Tyr Cys Asp Ser Gln
Cys Pro Arg Asp Leu Lys Phe 170 175 180 atc aac ggt cag gcc aac gtc
gag ggt tgg cag cct tcg tcg aac gat 672Ile Asn Gly Gln Ala Asn Val
Glu Gly Trp Gln Pro Ser Ser Asn Asp 185 190 195 gcc aac gca ggt acc
ggc aac cac ggt tcc tgt tgt gcc gaa atg gac 720Ala Asn Ala Gly Thr
Gly Asn His Gly Ser Cys Cys Ala Glu Met Asp 200 205 210 att tgg gaa
gcg aac tcg atc tcg acg gcg ttc act cct cac ccg tgt 768Ile Trp Glu
Ala Asn Ser Ile Ser Thr Ala Phe Thr Pro His Pro Cys 215 220 225 230
gat aca ccc gga cag gtg atg tgt aca ggc gac gcc tgt ggc gga acc
816Asp Thr Pro Gly Gln Val Met Cys Thr Gly Asp Ala Cys Gly Gly Thr
235 240 245 tac tcg tcg gat cga tat ggc ggt acg tgt gac ccc gac ggc
tgt gac 864Tyr Ser Ser Asp Arg Tyr Gly Gly Thr Cys Asp Pro Asp Gly
Cys Asp 250 255 260 ttc aac tcc ttc agg cag ggc aac aaa aca ttc tat
gga cct ggc atg 912Phe Asn Ser Phe Arg Gln Gly Asn Lys Thr Phe Tyr
Gly Pro Gly Met 265 270 275 acg gtg gat aca aag tcg aaa ttc aca gtc
gtc act cag ttc atc acc 960Thr Val Asp Thr Lys Ser Lys Phe Thr Val
Val Thr Gln Phe Ile Thr 280 285 290 gac gat ggt acg tcc tcg ggt acc
ttg aag gag atc aaa agg ttc tat 1008Asp Asp Gly Thr Ser Ser Gly Thr
Leu Lys Glu Ile Lys Arg Phe Tyr 295 300 305 310 gtc cag aac gga aag
gtc atc ccg aac tcg gag tcc acg tgg aca gga 1056Val Gln Asn Gly Lys
Val Ile Pro Asn Ser Glu Ser Thr Trp Thr Gly 315 320 325 gtg tcg ggt
aac tcc atc act acg gag tat tgt aca gcc cag aag tcg 1104Val Ser Gly
Asn Ser Ile Thr Thr Glu Tyr Cys Thr Ala Gln Lys Ser 330 335 340 ctc
ttc cag gat cag aac gtc ttc gag aaa cat gga ggc ttg gaa gga 1152Leu
Phe Gln Asp Gln Asn Val Phe Glu Lys His Gly Gly Leu Glu Gly 345 350
355 atg ggt gcc gca ttg gcc cag ggt atg gtc ctc gtc atg tcc ttg tgg
1200Met Gly Ala Ala Leu Ala Gln Gly Met Val Leu Val Met Ser Leu Trp
360 365 370 gac gac cac tcg gcc aac atg ctc tgg ttg gat tcc aac tac
ccc acc 1248Asp Asp His Ser Ala Asn Met Leu Trp Leu Asp Ser Asn Tyr
Pro Thr 375 380 385 390 act gcc tcg tcc acg aca ccg ggt gtc gca cgc
gga act tgt gat atc 1296Thr Ala Ser Ser Thr Thr Pro Gly Val Ala Arg
Gly Thr Cys Asp Ile 395 400 405 tcc tcg gga gtg cct gca gac gtc gag
gcg aac cat ccc gac gcc tac 1344Ser Ser Gly Val Pro Ala Asp Val Glu
Ala Asn His Pro Asp Ala Tyr 410 415 420 gtg gtc tac tcg aac att aag
gtg gga ccc atc ggt tcg aca ttc aac 1392Val Val Tyr Ser Asn Ile Lys
Val Gly Pro Ile Gly Ser Thr Phe Asn 425 430 435 tcc gga ggc tcg aac
cct gga ggc gga acg acc act act aca acg act 1440Ser Gly Gly Ser Asn
Pro Gly Gly Gly Thr Thr Thr Thr Thr Thr Thr 440 445 450 cag ccg aca
aca aca act acc aca gca ggc aac cct gga ggt aca ggt 1488Gln Pro Thr
Thr Thr Thr Thr Thr Ala Gly Asn Pro Gly Gly Thr Gly 455 460 465 470
gtg gcc cag cac tgg gga cag tgt ggc ggt atc gga tgg aca gga cct
1536Val Ala Gln His Trp Gly Gln Cys Gly Gly Ile Gly Trp Thr Gly Pro
475 480 485 act act tgt gca tcg cct tat acc tgt cag aaa ttg aac gac
tac tac 1584Thr Thr Cys Ala Ser Pro Tyr Thr Cys Gln Lys Leu Asn Asp
Tyr Tyr 490 495 500 tcg cag tgt ttg taa 1599Ser Gln Cys Leu 505
42532PRTAspergillus fumigatus 42Met Leu Ala Ser Thr Phe Ser Tyr Arg
Met Tyr Lys Thr Ala Leu Ile -25 -20 -15 Leu Ala Ala Leu Leu Gly Ser
Gly Gln Ala Gln Gln Val Gly Thr Ser -10 -5 -1 1 5 Gln Ala Glu Val
His Pro Ser Met Thr Trp Gln Ser Cys Thr Ala Gly 10 15 20 Gly Ser
Cys Thr Thr Asn Asn Gly Lys Val Val Ile Asp Ala Asn Trp 25 30 35
Arg Trp Val His Lys Thr Gly Asp Tyr Thr Asn Cys Tyr Thr Gly Asn 40
45 50 Thr Trp Asp Thr Thr Ile Cys Pro Asp Asp Ala Thr Cys Ala Ser
Asn 55 60 65 70 Cys Ala Leu Glu Gly Ala Asn Tyr Glu Ser Thr Tyr Gly
Val Thr Ala 75 80 85 Ser Gly Asn Ser Leu Arg Leu Asn Phe Val Thr
Thr Ser Gln Gln Lys 90 95 100 Asn Ile Gly Ser Arg Leu Tyr Met Met
Lys Asp Asp Ser Thr Tyr Glu 105 110 115 Met Phe Lys Leu Leu Asn Gln
Glu Phe Thr Phe Asp Val Asp Val Ser 120 125 130 Asn Leu Pro Cys Gly
Leu Asn Gly Ala Leu Tyr Phe Val Ala Met Asp 135 140 145 150 Ala Asp
Gly Gly Met Ser Lys Tyr Pro Thr Asn Lys Ala Gly Ala Lys 155 160 165
Tyr Gly Thr Gly Tyr Cys Asp Ser Gln Cys Pro Arg Asp Leu Lys Phe 170
175 180 Ile Asn Gly Gln Ala Asn Val Glu Gly Trp Gln Pro Ser Ser Asn
Asp 185 190 195 Ala Asn Ala Gly Thr Gly Asn His Gly Ser Cys Cys Ala
Glu Met Asp 200 205 210 Ile Trp Glu Ala Asn Ser Ile Ser Thr Ala Phe
Thr Pro His Pro Cys 215 220 225 230 Asp Thr Pro Gly Gln Val Met Cys
Thr Gly Asp Ala Cys Gly Gly Thr 235 240 245 Tyr Ser Ser Asp Arg Tyr
Gly Gly Thr Cys Asp Pro Asp Gly Cys Asp 250 255 260 Phe Asn Ser Phe
Arg Gln Gly Asn Lys Thr Phe Tyr Gly Pro Gly Met 265 270 275 Thr Val
Asp Thr Lys Ser Lys Phe Thr Val Val Thr Gln Phe Ile Thr 280 285 290
Asp Asp Gly Thr Ser Ser Gly Thr Leu Lys Glu Ile Lys Arg Phe Tyr 295
300 305 310 Val Gln Asn Gly Lys Val Ile Pro Asn Ser Glu Ser Thr Trp
Thr Gly 315 320 325 Val Ser Gly Asn Ser Ile Thr Thr Glu Tyr Cys Thr
Ala Gln Lys Ser 330 335 340 Leu Phe Gln Asp Gln Asn Val Phe Glu Lys
His Gly Gly Leu Glu Gly 345 350 355 Met Gly Ala Ala Leu Ala Gln Gly
Met Val Leu Val Met Ser Leu Trp 360 365 370 Asp Asp His Ser Ala Asn
Met Leu Trp Leu Asp Ser Asn Tyr Pro Thr 375 380 385 390 Thr Ala Ser
Ser Thr Thr Pro Gly Val Ala Arg Gly Thr Cys Asp Ile 395 400 405 Ser
Ser Gly Val Pro Ala Asp Val Glu Ala Asn His Pro Asp Ala Tyr 410 415
420 Val Val Tyr Ser Asn Ile Lys Val Gly Pro Ile Gly Ser Thr Phe Asn
425 430 435 Ser Gly Gly Ser Asn Pro Gly Gly Gly Thr Thr Thr Thr Thr
Thr Thr 440 445 450 Gln Pro Thr Thr Thr Thr Thr Thr Ala Gly Asn Pro
Gly Gly Thr Gly 455 460 465 470 Val Ala Gln His Trp Gly Gln Cys Gly
Gly Ile Gly Trp Thr Gly Pro 475 480 485 Thr Thr Cys Ala Ser Pro Tyr
Thr Cys Gln Lys Leu Asn Asp Tyr Tyr 490 495 500 Ser Gln Cys Leu 505
4343DNAArtificial SequenceSYNTHETIC PRIMER 43gtacaggtgt ggcccagcac
tggggacagt gtggcggtat cgg 434431DNAArtificial SequenceSYNTHETIC
PRIMER 44gtgctgggcc acacctgtac ctccagggtt g 31451599DNAAspergillus
fumigatussig_peptide(1)..(26)CDS(1)..(1596)mat_peptide(79)..(1596)
45atg ttg gcc tcc acg ttc tcc tat cgc atg tac aaa aca gcg ctc atc
48Met Leu Ala Ser Thr Phe Ser Tyr Arg Met Tyr Lys Thr Ala Leu Ile
-25 -20 -15 ttg gca gcc ctc ttg ggc tcg gga cag gca cag cag gtc gga
acc tcg 96Leu Ala Ala Leu Leu Gly Ser Gly Gln Ala Gln Gln Val Gly
Thr Ser -10 -5 -1 1 5 cag gcc gag gtc cat cct tcc atg acg tgg cag
tcg tgt aca gcg ggt 144Gln Ala Glu Val His Pro Ser Met Thr Trp Gln
Ser Cys Thr Ala Gly 10 15 20 ggt tcg tgt acc aca aac aac ggt aaa
gtc gtg atc gat gca aac tgg 192Gly Ser Cys Thr Thr Asn Asn Gly Lys
Val Val Ile Asp Ala Asn Trp 25 30 35 agg tgg gtg cac aag aca ggc
gac tac acc aac tgt tac aca ggc aac 240Arg Trp Val His Lys Thr Gly
Asp Tyr Thr Asn Cys Tyr Thr Gly Asn 40 45 50 aca tgg gat aca acc
atc tgt ccc gac gat gcc act tgt gca tcc aac 288Thr Trp Asp Thr Thr
Ile Cys Pro Asp Asp Ala Thr Cys Ala Ser Asn 55 60 65 70 tgt gca ctc
gag ggt gcc aac tat gag tcg acg tac gga gtg acc gcc 336Cys Ala Leu
Glu Gly Ala Asn Tyr Glu Ser Thr Tyr Gly Val Thr Ala 75 80 85 tcc
gga aac tcg ctc agg ctc aac ttc gtc aca act tcc cag cag aag 384Ser
Gly Asn Ser Leu Arg Leu Asn Phe Val Thr Thr Ser Gln Gln Lys 90 95
100 aac atc ggc tcg cgg ttg tat atg atg aaa gac gat tcc act tac gag
432Asn Ile Gly Ser Arg Leu Tyr Met Met Lys Asp Asp Ser Thr Tyr Glu
105 110 115 atg ttc aag ctc ctc aac cag gaa ttc act ttc gat gtc gac
gtc tcc 480Met Phe Lys Leu Leu Asn Gln Glu Phe Thr Phe Asp Val Asp
Val Ser 120 125 130 aac ctc cct tgt ggc ttg aac gga gcg ctc tac ttc
gtc gcc atg gat 528Asn Leu Pro Cys Gly Leu Asn Gly Ala Leu Tyr Phe
Val Ala Met Asp 135 140 145 150 gcg gat gga ggc atg tcc aag tat cct
acc aac aaa gca gga gcc aag 576Ala Asp Gly Gly Met Ser Lys Tyr Pro
Thr Asn Lys Ala Gly Ala Lys 155 160 165 tat ggt aca ggt tac tgt gat
tcc cag tgt ccc agg gat ctc aag ttc 624Tyr Gly Thr Gly Tyr Cys Asp
Ser Gln Cys Pro Arg Asp Leu Lys Phe 170 175 180 atc aac ggt cag gcc
aac gtc gag ggt tgg cag cct tcg tcg aac gat 672Ile Asn Gly Gln Ala
Asn Val Glu Gly Trp Gln Pro Ser Ser Asn Asp 185 190 195 gcc aac gca
ggt acc ggc aac cac ggt tcc tgt tgt gcc gaa atg gac 720Ala Asn Ala
Gly Thr Gly Asn His Gly Ser Cys Cys Ala Glu Met Asp 200 205 210 att
tgg gaa gcg aac tcg atc tcg acg gcg ttc act cct cac ccg tgt 768Ile
Trp Glu Ala Asn Ser Ile Ser Thr Ala Phe Thr Pro His Pro Cys 215 220
225 230 gat aca ccc gga cag gtg atg tgt aca ggc gac gcc tgt ggc gga
acc 816Asp Thr Pro Gly Gln Val Met Cys Thr Gly Asp Ala Cys Gly Gly
Thr 235 240 245 tac tcg tcg gat cga tat ggc ggt acg tgt gac ccc gac
ggc tgt gac 864Tyr Ser Ser Asp Arg Tyr Gly Gly Thr Cys Asp Pro Asp
Gly Cys Asp 250 255 260 ttc aac cct ttc agg cag ggc aac aaa aca ttc
tat gga cct ggc atg 912Phe Asn Pro Phe Arg Gln Gly Asn Lys Thr Phe
Tyr Gly Pro Gly Met 265 270 275 acg gtg gat aca aag tcg aaa ttc aca
gtc gtc act cag ttc atc acc 960Thr Val Asp Thr Lys Ser Lys Phe Thr
Val Val Thr Gln Phe Ile Thr 280 285 290 gac gat ggt acg tcc tcg ggt
acc ttg aag gag atc aaa agg ttc tat 1008Asp Asp Gly Thr Ser Ser Gly
Thr Leu Lys Glu Ile Lys Arg Phe Tyr 295 300 305 310 gtc cag aac gga
aag gtc atc ccg aac tcg gag tcc acg tgg aca gga 1056Val Gln Asn Gly
Lys Val Ile Pro Asn Ser Glu Ser Thr Trp Thr Gly 315 320 325 gtg tcg
ggt aac tcc atc act acg gag tat tgt aca gcc cag aag tcg 1104Val Ser
Gly Asn Ser Ile Thr Thr Glu Tyr Cys Thr Ala Gln Lys Ser 330 335 340
ctc ttc cag gat cag aac gtc ttc gag aaa cat gga ggc ttg gaa gga
1152Leu Phe Gln Asp Gln Asn Val Phe Glu Lys His Gly Gly Leu Glu Gly
345 350 355 atg ggt gcc gca ttg gcc cag ggt atg gtc ctc gtc atg tcc
ttg tgg 1200Met Gly Ala Ala Leu Ala Gln Gly Met Val Leu Val Met Ser
Leu Trp 360 365 370 gac gac cac tcg gcc aac atg ctc tgg ttg gat tcc
aac tac ccc acc 1248Asp Asp His Ser Ala Asn Met Leu Trp Leu Asp Ser
Asn Tyr Pro Thr 375 380 385 390 act gcc tcg tcc acg aca ccg ggt gtc
gca cgc gga act tgt gat atc 1296Thr Ala Ser Ser Thr Thr Pro Gly Val
Ala Arg Gly Thr Cys Asp Ile 395 400 405 tcc tcg gga gtg cct gca gac
gtc gag gcg aac cat ccc gac gcc tac 1344Ser Ser Gly Val Pro Ala Asp
Val Glu Ala Asn His Pro Asp Ala Tyr 410 415 420 gtg gtc tac tcg aac
att aag gtg gga ccc atc ggt tcg aca ttc aac 1392Val Val Tyr Ser Asn
Ile Lys Val Gly Pro Ile Gly Ser Thr Phe Asn 425 430 435 tcc gga ggc
tcg aac cct gga ggc gga acg acc act act aca acg act 1440Ser Gly Gly
Ser Asn Pro Gly Gly Gly Thr Thr Thr Thr Thr Thr Thr 440 445 450 cag
ccg aca aca aca act acc aca gca ggc aac cct gga ggt aca ggt 1488Gln
Pro Thr Thr Thr Thr Thr Thr Ala Gly Asn Pro Gly Gly Thr Gly 455 460
465 470 gtg gcc cag cac tgg gga cag tgt ggc ggt atc gga tgg aca gga
cct 1536Val Ala Gln His Trp Gly Gln Cys Gly Gly Ile Gly Trp Thr Gly
Pro 475 480 485 act act tgt gca tcg cct tat acc tgt cag aaa ttg aac
gac tac tac 1584Thr Thr Cys Ala Ser Pro Tyr Thr Cys Gln Lys Leu Asn
Asp Tyr Tyr 490 495 500 tcg cag tgt ttg taa 1599Ser Gln Cys Leu 505
46532PRTAspergillus fumigatus 46Met Leu Ala Ser Thr Phe Ser Tyr Arg
Met Tyr Lys Thr Ala Leu Ile -25 -20 -15 Leu Ala Ala Leu Leu Gly Ser
Gly Gln Ala Gln Gln Val Gly Thr Ser -10 -5 -1 1 5 Gln
Ala Glu Val His Pro Ser Met Thr Trp Gln Ser Cys Thr Ala Gly 10 15
20 Gly Ser Cys Thr Thr Asn Asn Gly Lys Val Val Ile Asp Ala Asn Trp
25 30 35 Arg Trp Val His Lys Thr Gly Asp Tyr Thr Asn Cys Tyr Thr
Gly Asn 40 45 50 Thr Trp Asp Thr Thr Ile Cys Pro Asp Asp Ala Thr
Cys Ala Ser Asn 55 60 65 70 Cys Ala Leu Glu Gly Ala Asn Tyr Glu Ser
Thr Tyr Gly Val Thr Ala 75 80 85 Ser Gly Asn Ser Leu Arg Leu Asn
Phe Val Thr Thr Ser Gln Gln Lys 90 95 100 Asn Ile Gly Ser Arg Leu
Tyr Met Met Lys Asp Asp Ser Thr Tyr Glu 105 110 115 Met Phe Lys Leu
Leu Asn Gln Glu Phe Thr Phe Asp Val Asp Val Ser 120 125 130 Asn Leu
Pro Cys Gly Leu Asn Gly Ala Leu Tyr Phe Val Ala Met Asp 135 140 145
150 Ala Asp Gly Gly Met Ser Lys Tyr Pro Thr Asn Lys Ala Gly Ala Lys
155 160 165 Tyr Gly Thr Gly Tyr Cys Asp Ser Gln Cys Pro Arg Asp Leu
Lys Phe 170 175 180 Ile Asn Gly Gln Ala Asn Val Glu Gly Trp Gln Pro
Ser Ser Asn Asp 185 190 195 Ala Asn Ala Gly Thr Gly Asn His Gly Ser
Cys Cys Ala Glu Met Asp 200 205 210 Ile Trp Glu Ala Asn Ser Ile Ser
Thr Ala Phe Thr Pro His Pro Cys 215 220 225 230 Asp Thr Pro Gly Gln
Val Met Cys Thr Gly Asp Ala Cys Gly Gly Thr 235 240 245 Tyr Ser Ser
Asp Arg Tyr Gly Gly Thr Cys Asp Pro Asp Gly Cys Asp 250 255 260 Phe
Asn Pro Phe Arg Gln Gly Asn Lys Thr Phe Tyr Gly Pro Gly Met 265 270
275 Thr Val Asp Thr Lys Ser Lys Phe Thr Val Val Thr Gln Phe Ile Thr
280 285 290 Asp Asp Gly Thr Ser Ser Gly Thr Leu Lys Glu Ile Lys Arg
Phe Tyr 295 300 305 310 Val Gln Asn Gly Lys Val Ile Pro Asn Ser Glu
Ser Thr Trp Thr Gly 315 320 325 Val Ser Gly Asn Ser Ile Thr Thr Glu
Tyr Cys Thr Ala Gln Lys Ser 330 335 340 Leu Phe Gln Asp Gln Asn Val
Phe Glu Lys His Gly Gly Leu Glu Gly 345 350 355 Met Gly Ala Ala Leu
Ala Gln Gly Met Val Leu Val Met Ser Leu Trp 360 365 370 Asp Asp His
Ser Ala Asn Met Leu Trp Leu Asp Ser Asn Tyr Pro Thr 375 380 385 390
Thr Ala Ser Ser Thr Thr Pro Gly Val Ala Arg Gly Thr Cys Asp Ile 395
400 405 Ser Ser Gly Val Pro Ala Asp Val Glu Ala Asn His Pro Asp Ala
Tyr 410 415 420 Val Val Tyr Ser Asn Ile Lys Val Gly Pro Ile Gly Ser
Thr Phe Asn 425 430 435 Ser Gly Gly Ser Asn Pro Gly Gly Gly Thr Thr
Thr Thr Thr Thr Thr 440 445 450 Gln Pro Thr Thr Thr Thr Thr Thr Ala
Gly Asn Pro Gly Gly Thr Gly 455 460 465 470 Val Ala Gln His Trp Gly
Gln Cys Gly Gly Ile Gly Trp Thr Gly Pro 475 480 485 Thr Thr Cys Ala
Ser Pro Tyr Thr Cys Gln Lys Leu Asn Asp Tyr Tyr 490 495 500 Ser Gln
Cys Leu 505 471599DNAAspergillus
fumigatussig_peptide(1)..(26)CDS(1)..(1596)mat_peptide(79)..(1596)
47atg ttg gcc tcc acg ttc tcc tat cgc atg tac aaa aca gcg ctc atc
48Met Leu Ala Ser Thr Phe Ser Tyr Arg Met Tyr Lys Thr Ala Leu Ile
-25 -20 -15 ttg gca gcc ctc ttg ggc tcg gga cag gca cag cag gtc gga
acc tcg 96Leu Ala Ala Leu Leu Gly Ser Gly Gln Ala Gln Gln Val Gly
Thr Ser -10 -5 -1 1 5 cag gcc gag gtc cat cct tcc atg acg tgg cag
tcg tgt aca gcg ggt 144Gln Ala Glu Val His Pro Ser Met Thr Trp Gln
Ser Cys Thr Ala Gly 10 15 20 ggt tcg tgt acc aca aac aac ggt aaa
gtc gtg atc gat gca aac tgg 192Gly Ser Cys Thr Thr Asn Asn Gly Lys
Val Val Ile Asp Ala Asn Trp 25 30 35 agg tgg gtg cac aag aca ggc
gac tac acc aac tgt tac aca ggc aac 240Arg Trp Val His Lys Thr Gly
Asp Tyr Thr Asn Cys Tyr Thr Gly Asn 40 45 50 aca tgg gat aca acc
atc tgt ccc gac gat gcc act tgt gca tcc aac 288Thr Trp Asp Thr Thr
Ile Cys Pro Asp Asp Ala Thr Cys Ala Ser Asn 55 60 65 70 tgt gca ctc
gag ggt gcc aac tat gag tcg acg tac gga gtg acc gcc 336Cys Ala Leu
Glu Gly Ala Asn Tyr Glu Ser Thr Tyr Gly Val Thr Ala 75 80 85 tcc
gga aac tcg ctc agg ctc aac ttc gtc aca act tcc cag cag aag 384Ser
Gly Asn Ser Leu Arg Leu Asn Phe Val Thr Thr Ser Gln Gln Lys 90 95
100 aac atc ggc tcg cgg ttg tat atg atg aaa gac gat tcc act tac gag
432Asn Ile Gly Ser Arg Leu Tyr Met Met Lys Asp Asp Ser Thr Tyr Glu
105 110 115 atg ttc aag ctc ctc aac cag gaa ttc act ttc gat gtc gac
gtc tcc 480Met Phe Lys Leu Leu Asn Gln Glu Phe Thr Phe Asp Val Asp
Val Ser 120 125 130 aac ctc cct tgt ggc ttg aac gga gcg ctc tac ttc
gtc gcc atg gat 528Asn Leu Pro Cys Gly Leu Asn Gly Ala Leu Tyr Phe
Val Ala Met Asp 135 140 145 150 gcg gat gga ggc atg tcc aag tat cct
acc aac aaa gca gga gcc aag 576Ala Asp Gly Gly Met Ser Lys Tyr Pro
Thr Asn Lys Ala Gly Ala Lys 155 160 165 tat ggt aca ggt tac tgt gat
tcc cag tgt ccc agg gat ctc aag ttc 624Tyr Gly Thr Gly Tyr Cys Asp
Ser Gln Cys Pro Arg Asp Leu Lys Phe 170 175 180 atc aac ggt cag gcc
aac gtc gag ggt tgg cag cct tcg tcg aac gat 672Ile Asn Gly Gln Ala
Asn Val Glu Gly Trp Gln Pro Ser Ser Asn Asp 185 190 195 gcc aac gca
ggt acc ggc aac cac ggt tcc tgt tgt gcc gaa atg gac 720Ala Asn Ala
Gly Thr Gly Asn His Gly Ser Cys Cys Ala Glu Met Asp 200 205 210 att
tgg gaa gcg aac tcg atc tcg acg gcg ttc act cct cac ccg tgt 768Ile
Trp Glu Ala Asn Ser Ile Ser Thr Ala Phe Thr Pro His Pro Cys 215 220
225 230 gat aca ccc gga cag gtg atg tgt aca ggc gac gcc tgt ggc gga
acc 816Asp Thr Pro Gly Gln Val Met Cys Thr Gly Asp Ala Cys Gly Gly
Thr 235 240 245 tac tcg tcg gat cga tat ggc ggt acg tgt gac ccc gac
ggc tgt gac 864Tyr Ser Ser Asp Arg Tyr Gly Gly Thr Cys Asp Pro Asp
Gly Cys Asp 250 255 260 ttc aac cct ttc agg cag ggc aac aaa aca ttc
tat gga cct ggc atg 912Phe Asn Pro Phe Arg Gln Gly Asn Lys Thr Phe
Tyr Gly Pro Gly Met 265 270 275 acg gtg gat aca aag tcg aaa ttc aca
gtc gtc act cag ttc atc acc 960Thr Val Asp Thr Lys Ser Lys Phe Thr
Val Val Thr Gln Phe Ile Thr 280 285 290 gac gat ggt acg tcc tcg ggt
acc ttg aag gag atc aaa agg ttc tat 1008Asp Asp Gly Thr Ser Ser Gly
Thr Leu Lys Glu Ile Lys Arg Phe Tyr 295 300 305 310 gtc cag aac gga
aag gtc atc ccg aac tcg gag tcc acg tgg aca gga 1056Val Gln Asn Gly
Lys Val Ile Pro Asn Ser Glu Ser Thr Trp Thr Gly 315 320 325 gtg tcg
ggt aac tcc atc act acg gag tat tgt aca gcc cag aag tcg 1104Val Ser
Gly Asn Ser Ile Thr Thr Glu Tyr Cys Thr Ala Gln Lys Ser 330 335 340
ctc ttc cag gat cag aac gtc ttc gag aaa cat gga ggc ttg gaa gga
1152Leu Phe Gln Asp Gln Asn Val Phe Glu Lys His Gly Gly Leu Glu Gly
345 350 355 atg ggt gcc gca ttg gcc cag ggt atg gtc ctc gtc atg tcc
ttg tgg 1200Met Gly Ala Ala Leu Ala Gln Gly Met Val Leu Val Met Ser
Leu Trp 360 365 370 gac gac cac tcg gcc aac atg ctc tgg ttg gat tcc
aac tac ccc acc 1248Asp Asp His Ser Ala Asn Met Leu Trp Leu Asp Ser
Asn Tyr Pro Thr 375 380 385 390 gat gcc gat cct acg aca ccg ggt gtc
gca cgc gga act tgt gat atc 1296Asp Ala Asp Pro Thr Thr Pro Gly Val
Ala Arg Gly Thr Cys Asp Ile 395 400 405 tcc tcg gga gtg cct gca gac
gtc gag gcg aac cat ccc gac gcc tac 1344Ser Ser Gly Val Pro Ala Asp
Val Glu Ala Asn His Pro Asp Ala Tyr 410 415 420 gtg gtc tac tcg aac
att aag gtg gga ccc atc ggt tcg aca ttc aac 1392Val Val Tyr Ser Asn
Ile Lys Val Gly Pro Ile Gly Ser Thr Phe Asn 425 430 435 tcc gga ggc
tcg aac cct gga ggc gga acg acc act act aca acg act 1440Ser Gly Gly
Ser Asn Pro Gly Gly Gly Thr Thr Thr Thr Thr Thr Thr 440 445 450 cag
ccg aca aca aca act acc aca gca ggc aac cct gga ggt aca ggt 1488Gln
Pro Thr Thr Thr Thr Thr Thr Ala Gly Asn Pro Gly Gly Thr Gly 455 460
465 470 gtg gcc cag cac tgg gga cag tgt ggc ggt atc gga tgg aca gga
cct 1536Val Ala Gln His Trp Gly Gln Cys Gly Gly Ile Gly Trp Thr Gly
Pro 475 480 485 act act tgt gca tcg cct tat acc tgt cag aaa ttg aac
gac tac tac 1584Thr Thr Cys Ala Ser Pro Tyr Thr Cys Gln Lys Leu Asn
Asp Tyr Tyr 490 495 500 tcg cag tgt ttg taa 1599Ser Gln Cys Leu 505
48532PRTAspergillus fumigatus 48Met Leu Ala Ser Thr Phe Ser Tyr Arg
Met Tyr Lys Thr Ala Leu Ile -25 -20 -15 Leu Ala Ala Leu Leu Gly Ser
Gly Gln Ala Gln Gln Val Gly Thr Ser -10 -5 -1 1 5 Gln Ala Glu Val
His Pro Ser Met Thr Trp Gln Ser Cys Thr Ala Gly 10 15 20 Gly Ser
Cys Thr Thr Asn Asn Gly Lys Val Val Ile Asp Ala Asn Trp 25 30 35
Arg Trp Val His Lys Thr Gly Asp Tyr Thr Asn Cys Tyr Thr Gly Asn 40
45 50 Thr Trp Asp Thr Thr Ile Cys Pro Asp Asp Ala Thr Cys Ala Ser
Asn 55 60 65 70 Cys Ala Leu Glu Gly Ala Asn Tyr Glu Ser Thr Tyr Gly
Val Thr Ala 75 80 85 Ser Gly Asn Ser Leu Arg Leu Asn Phe Val Thr
Thr Ser Gln Gln Lys 90 95 100 Asn Ile Gly Ser Arg Leu Tyr Met Met
Lys Asp Asp Ser Thr Tyr Glu 105 110 115 Met Phe Lys Leu Leu Asn Gln
Glu Phe Thr Phe Asp Val Asp Val Ser 120 125 130 Asn Leu Pro Cys Gly
Leu Asn Gly Ala Leu Tyr Phe Val Ala Met Asp 135 140 145 150 Ala Asp
Gly Gly Met Ser Lys Tyr Pro Thr Asn Lys Ala Gly Ala Lys 155 160 165
Tyr Gly Thr Gly Tyr Cys Asp Ser Gln Cys Pro Arg Asp Leu Lys Phe 170
175 180 Ile Asn Gly Gln Ala Asn Val Glu Gly Trp Gln Pro Ser Ser Asn
Asp 185 190 195 Ala Asn Ala Gly Thr Gly Asn His Gly Ser Cys Cys Ala
Glu Met Asp 200 205 210 Ile Trp Glu Ala Asn Ser Ile Ser Thr Ala Phe
Thr Pro His Pro Cys 215 220 225 230 Asp Thr Pro Gly Gln Val Met Cys
Thr Gly Asp Ala Cys Gly Gly Thr 235 240 245 Tyr Ser Ser Asp Arg Tyr
Gly Gly Thr Cys Asp Pro Asp Gly Cys Asp 250 255 260 Phe Asn Pro Phe
Arg Gln Gly Asn Lys Thr Phe Tyr Gly Pro Gly Met 265 270 275 Thr Val
Asp Thr Lys Ser Lys Phe Thr Val Val Thr Gln Phe Ile Thr 280 285 290
Asp Asp Gly Thr Ser Ser Gly Thr Leu Lys Glu Ile Lys Arg Phe Tyr 295
300 305 310 Val Gln Asn Gly Lys Val Ile Pro Asn Ser Glu Ser Thr Trp
Thr Gly 315 320 325 Val Ser Gly Asn Ser Ile Thr Thr Glu Tyr Cys Thr
Ala Gln Lys Ser 330 335 340 Leu Phe Gln Asp Gln Asn Val Phe Glu Lys
His Gly Gly Leu Glu Gly 345 350 355 Met Gly Ala Ala Leu Ala Gln Gly
Met Val Leu Val Met Ser Leu Trp 360 365 370 Asp Asp His Ser Ala Asn
Met Leu Trp Leu Asp Ser Asn Tyr Pro Thr 375 380 385 390 Asp Ala Asp
Pro Thr Thr Pro Gly Val Ala Arg Gly Thr Cys Asp Ile 395 400 405 Ser
Ser Gly Val Pro Ala Asp Val Glu Ala Asn His Pro Asp Ala Tyr 410 415
420 Val Val Tyr Ser Asn Ile Lys Val Gly Pro Ile Gly Ser Thr Phe Asn
425 430 435 Ser Gly Gly Ser Asn Pro Gly Gly Gly Thr Thr Thr Thr Thr
Thr Thr 440 445 450 Gln Pro Thr Thr Thr Thr Thr Thr Ala Gly Asn Pro
Gly Gly Thr Gly 455 460 465 470 Val Ala Gln His Trp Gly Gln Cys Gly
Gly Ile Gly Trp Thr Gly Pro 475 480 485 Thr Thr Cys Ala Ser Pro Tyr
Thr Cys Gln Lys Leu Asn Asp Tyr Tyr 490 495 500 Ser Gln Cys Leu 505
4954DNAArtificial SequenceSYNTHETIC PRIMER 49gttggattcc aactacccca
ccgatgccga tcctacgaca ccgggtgtcg cacg 545034DNAArtificial
SequenceSYNTHETIC PRIMER 50ggtggggtag ttggaatcca accagagcat gttg
34511705DNAArtificial
SequenceSyntheticexon(1)..(603)sig_peptide(1)..(18)CDS(1)..(603)mat_pepti-
de(55)..(1702)Intron(604)..(667)exon(668)..(1235)CDS(668)..(1235)Intron(12-
36)..(1310)exon(1311)..(1702)CDS(1311)..(1702) 51atg ttt cga cgg
gct ctt ttc ctg tcc tct tcc gcc ttc ctt gct gtc 48Met Phe Arg Arg
Ala Leu Phe Leu Ser Ser Ser Ala Phe Leu Ala Val -15 -10 -5 aaa gcc
cag cag atc ggc acg gtc agt ccg gag aac cat ccg ccc ctg 96Lys Ala
Gln Gln Ile Gly Thr Val Ser Pro Glu Asn His Pro Pro Leu -1 1 5 10
gca tgg gag cag tgc act gcc cct ggg agt tgc acg act gtg aat ggt
144Ala Trp Glu Gln Cys Thr Ala Pro Gly Ser Cys Thr Thr Val Asn Gly
15 20 25 30 gcg gtc gtc ctt gat gcg aac tgg cgt tgg gtc cac aat gtt
ggg gga 192Ala Val Val Leu Asp Ala Asn Trp Arg Trp Val His Asn Val
Gly Gly 35 40 45 tac acc aac tgc tac act ggc aat acc tgg gac acc
acg tac tgc cct 240Tyr Thr Asn Cys Tyr Thr Gly Asn Thr Trp Asp Thr
Thr Tyr Cys Pro 50 55 60 gac gac gtg acc tgc gca gag aat tgt gcg
ctg gat ggc gca gat tac 288Asp Asp Val Thr Cys Ala Glu Asn Cys Ala
Leu Asp Gly Ala Asp Tyr 65 70 75 gag ggc acc tac ggc gtg acc acc
tcg ggc agc tcc ctg aag ctc gat 336Glu Gly Thr Tyr Gly Val Thr Thr
Ser Gly Ser Ser Leu Lys Leu Asp 80 85 90 ttc gtc acc ggg tct aac
gtc gga tct cgt ctc tac ctg ttg gag aat 384Phe Val Thr Gly Ser Asn
Val Gly Ser Arg Leu Tyr Leu Leu Glu Asn 95 100 105 110 gat tcg acc
tat cag atc ttc aag ctt ctg aac cag gaa ttc acc ttt 432Asp Ser Thr
Tyr Gln Ile Phe Lys Leu Leu Asn Gln Glu Phe Thr Phe 115 120 125 gac
gtc gac gtt tcc aat ctt ccg tgc gga tta aac ggc gct ctg tac 480Asp
Val Asp Val Ser Asn Leu Pro Cys Gly Leu Asn Gly Ala Leu Tyr 130
135
140 ctt gtt acc atg gct gct gac ggc ggg gtg tct cag tac ccg aat aac
528Leu Val Thr Met Ala Ala Asp Gly Gly Val Ser Gln Tyr Pro Asn Asn
145 150 155 aag gcc ggc gca gcg tat gga acc ggt tat tgc gat tcc cag
tgt cca 576Lys Ala Gly Ala Ala Tyr Gly Thr Gly Tyr Cys Asp Ser Gln
Cys Pro 160 165 170 agg gac ttg aag ttt atc gat ggc cag gtatgtagag
ctgtaatcac 623Arg Asp Leu Lys Phe Ile Asp Gly Gln 175 180
ccatgttgtg aaatcactct cctactgaca tggtcgattt atag gcc aac gtt gag
679 Ala Asn Val Glu 185 ggc tgg cag ccg tct tcg aac aac gcc aat aca
ggt att ggc aac cat 727Gly Trp Gln Pro Ser Ser Asn Asn Ala Asn Thr
Gly Ile Gly Asn His 190 195 200 ggc tcc tgc tgt gcg gag atg gat atc
tgg gaa gcc aac agc atc tcc 775Gly Ser Cys Cys Ala Glu Met Asp Ile
Trp Glu Ala Asn Ser Ile Ser 205 210 215 aat gcg gtg act ccg cac cca
tgc gac aca ccc ggc cag aca atg tgc 823Asn Ala Val Thr Pro His Pro
Cys Asp Thr Pro Gly Gln Thr Met Cys 220 225 230 235 gag ggg aac gac
tgt ggt ggc acg tat tcc acc aat cgc tat gca ggc 871Glu Gly Asn Asp
Cys Gly Gly Thr Tyr Ser Thr Asn Arg Tyr Ala Gly 240 245 250 acc tgc
gat cct gac ggc tgc gac ttc aac ccc tac cgc atg ggc aac 919Thr Cys
Asp Pro Asp Gly Cys Asp Phe Asn Pro Tyr Arg Met Gly Asn 255 260 265
cat tct ttc tac ggc cct ggg gag att gtc gat act acc cag ccc ttc
967His Ser Phe Tyr Gly Pro Gly Glu Ile Val Asp Thr Thr Gln Pro Phe
270 275 280 act gtc gtg aca cag ttc ctt acc gat gat ggc acg gat act
ggc act 1015Thr Val Val Thr Gln Phe Leu Thr Asp Asp Gly Thr Asp Thr
Gly Thr 285 290 295 ctc agc gag atc aaa cgc ttc tac gtc caa aac ggg
aaa gtc att cct 1063Leu Ser Glu Ile Lys Arg Phe Tyr Val Gln Asn Gly
Lys Val Ile Pro 300 305 310 315 cag ccg aac tcc gac att gcc ggc gtg
act ggc aac tcg atc acc agc 1111Gln Pro Asn Ser Asp Ile Ala Gly Val
Thr Gly Asn Ser Ile Thr Ser 320 325 330 gag ttt tgc gat gcc cag aag
acg gct ttc ggc gac att aac aac ttt 1159Glu Phe Cys Asp Ala Gln Lys
Thr Ala Phe Gly Asp Ile Asn Asn Phe 335 340 345 gat aca cac ggc ggt
ctg gcc agt atg gga gct gcg ctg cag cag ggt 1207Asp Thr His Gly Gly
Leu Ala Ser Met Gly Ala Ala Leu Gln Gln Gly 350 355 360 atg gtt ctg
gtg atg agt ctg tgg gac g gtaggtcctt gggagacacc 1255Met Val Leu Val
Met Ser Leu Trp Asp 365 370 cggacgttct atatcaacca gaactgccag
aactgacgaa ttaaaacact tttag at 1312 Asp tac gcg gca aac atg ctg tgg
ttg gac agc att tat cca aca aat gca 1360Tyr Ala Ala Asn Met Leu Trp
Leu Asp Ser Ile Tyr Pro Thr Asn Ala 375 380 385 tct gct agc act cct
ggt gct gct cgt gga acc tgt tcg acg agc tcc 1408Ser Ala Ser Thr Pro
Gly Ala Ala Arg Gly Thr Cys Ser Thr Ser Ser 390 395 400 405 ggt gtc
cca tcg caa gtc gag tcg cag agc ccc aac gcc tac gtg acg 1456Gly Val
Pro Ser Gln Val Glu Ser Gln Ser Pro Asn Ala Tyr Val Thr 410 415 420
tac tcc aac att aaa gtt gga cca atc aac tcg acc ttc acc act tcg
1504Tyr Ser Asn Ile Lys Val Gly Pro Ile Asn Ser Thr Phe Thr Thr Ser
425 430 435 ggc tcg aac cct gga ggc gga acg acc act act aca acg act
cag ccg 1552Gly Ser Asn Pro Gly Gly Gly Thr Thr Thr Thr Thr Thr Thr
Gln Pro 440 445 450 aca aca aca act acc aca gca ggc aac cct gga ggt
aca ggt gtg gcc 1600Thr Thr Thr Thr Thr Thr Ala Gly Asn Pro Gly Gly
Thr Gly Val Ala 455 460 465 cag cac tac gga cag tgt ggc ggt atc gga
tgg aca gga cct act act 1648Gln His Tyr Gly Gln Cys Gly Gly Ile Gly
Trp Thr Gly Pro Thr Thr 470 475 480 485 tgt gca tcg cct tat acc tgt
cag aaa ttg aac gac tac tac tcg cag 1696Cys Ala Ser Pro Tyr Thr Cys
Gln Lys Leu Asn Asp Tyr Tyr Ser Gln 490 495 500 tgt ttg taa 1705Cys
Leu 52521PRTArtificial SequenceSynthetic Construct 52Met Phe Arg
Arg Ala Leu Phe Leu Ser Ser Ser Ala Phe Leu Ala Val -15 -10 -5 Lys
Ala Gln Gln Ile Gly Thr Val Ser Pro Glu Asn His Pro Pro Leu -1 1 5
10 Ala Trp Glu Gln Cys Thr Ala Pro Gly Ser Cys Thr Thr Val Asn Gly
15 20 25 30 Ala Val Val Leu Asp Ala Asn Trp Arg Trp Val His Asn Val
Gly Gly 35 40 45 Tyr Thr Asn Cys Tyr Thr Gly Asn Thr Trp Asp Thr
Thr Tyr Cys Pro 50 55 60 Asp Asp Val Thr Cys Ala Glu Asn Cys Ala
Leu Asp Gly Ala Asp Tyr 65 70 75 Glu Gly Thr Tyr Gly Val Thr Thr
Ser Gly Ser Ser Leu Lys Leu Asp 80 85 90 Phe Val Thr Gly Ser Asn
Val Gly Ser Arg Leu Tyr Leu Leu Glu Asn 95 100 105 110 Asp Ser Thr
Tyr Gln Ile Phe Lys Leu Leu Asn Gln Glu Phe Thr Phe 115 120 125 Asp
Val Asp Val Ser Asn Leu Pro Cys Gly Leu Asn Gly Ala Leu Tyr 130 135
140 Leu Val Thr Met Ala Ala Asp Gly Gly Val Ser Gln Tyr Pro Asn Asn
145 150 155 Lys Ala Gly Ala Ala Tyr Gly Thr Gly Tyr Cys Asp Ser Gln
Cys Pro 160 165 170 Arg Asp Leu Lys Phe Ile Asp Gly Gln Ala Asn Val
Glu Gly Trp Gln 175 180 185 190 Pro Ser Ser Asn Asn Ala Asn Thr Gly
Ile Gly Asn His Gly Ser Cys 195 200 205 Cys Ala Glu Met Asp Ile Trp
Glu Ala Asn Ser Ile Ser Asn Ala Val 210 215 220 Thr Pro His Pro Cys
Asp Thr Pro Gly Gln Thr Met Cys Glu Gly Asn 225 230 235 Asp Cys Gly
Gly Thr Tyr Ser Thr Asn Arg Tyr Ala Gly Thr Cys Asp 240 245 250 Pro
Asp Gly Cys Asp Phe Asn Pro Tyr Arg Met Gly Asn His Ser Phe 255 260
265 270 Tyr Gly Pro Gly Glu Ile Val Asp Thr Thr Gln Pro Phe Thr Val
Val 275 280 285 Thr Gln Phe Leu Thr Asp Asp Gly Thr Asp Thr Gly Thr
Leu Ser Glu 290 295 300 Ile Lys Arg Phe Tyr Val Gln Asn Gly Lys Val
Ile Pro Gln Pro Asn 305 310 315 Ser Asp Ile Ala Gly Val Thr Gly Asn
Ser Ile Thr Ser Glu Phe Cys 320 325 330 Asp Ala Gln Lys Thr Ala Phe
Gly Asp Ile Asn Asn Phe Asp Thr His 335 340 345 350 Gly Gly Leu Ala
Ser Met Gly Ala Ala Leu Gln Gln Gly Met Val Leu 355 360 365 Val Met
Ser Leu Trp Asp Asp Tyr Ala Ala Asn Met Leu Trp Leu Asp 370 375 380
Ser Ile Tyr Pro Thr Asn Ala Ser Ala Ser Thr Pro Gly Ala Ala Arg 385
390 395 Gly Thr Cys Ser Thr Ser Ser Gly Val Pro Ser Gln Val Glu Ser
Gln 400 405 410 Ser Pro Asn Ala Tyr Val Thr Tyr Ser Asn Ile Lys Val
Gly Pro Ile 415 420 425 430 Asn Ser Thr Phe Thr Thr Ser Gly Ser Asn
Pro Gly Gly Gly Thr Thr 435 440 445 Thr Thr Thr Thr Thr Gln Pro Thr
Thr Thr Thr Thr Thr Ala Gly Asn 450 455 460 Pro Gly Gly Thr Gly Val
Ala Gln His Tyr Gly Gln Cys Gly Gly Ile 465 470 475 Gly Trp Thr Gly
Pro Thr Thr Cys Ala Ser Pro Tyr Thr Cys Gln Lys 480 485 490 Leu Asn
Asp Tyr Tyr Ser Gln Cys Leu 495 500 5350DNAArtificial
SequenceSYNTHETIC PRIMER 53caatcaactc gaccttcacc acttcgggct
cgaaccctgg aggcggaacg 505434DNAArtificial SequenceSYNTHETIC PRIMER
54ctagatctcg agttacaaac actgcgagta gtag 345526DNAArtificial
SequenceSYNTHETIC PRIMER 55cgaagtggtg aaggtcgagt tgattg
26561705DNAArtificial
SequenceSyntheticexon(1)..(603)sig_peptide(1)..(18)CDS(1)..(603)mat_pepti-
de(55)..(1702)Intron(604)..(667)exon(668)..(1235)CDS(668)..(1235)Intron(12-
36)..(1310)exon(1311)..(1702)CDS(1311)..(1702) 56atg ttt cga cgg
gct ctt ttc ctg tcc tct tcc gcc ttc ctt gct gtc 48Met Phe Arg Arg
Ala Leu Phe Leu Ser Ser Ser Ala Phe Leu Ala Val -15 -10 -5 aaa gcc
cag cag atc ggc acg gtc agt ccg gag aac cat ccg ccc ctg 96Lys Ala
Gln Gln Ile Gly Thr Val Ser Pro Glu Asn His Pro Pro Leu -1 1 5 10
gca tgg gag cag tgc act gcc cct ggg agt tgc acg act gtg aat ggt
144Ala Trp Glu Gln Cys Thr Ala Pro Gly Ser Cys Thr Thr Val Asn Gly
15 20 25 30 gcg gtc gtc ctt gat gcg aac tgg cgt tgg gtc cac aat gtt
ggg gga 192Ala Val Val Leu Asp Ala Asn Trp Arg Trp Val His Asn Val
Gly Gly 35 40 45 tac acc aac tgc tac act ggc aat acc tgg gac acc
acg tac tgc cct 240Tyr Thr Asn Cys Tyr Thr Gly Asn Thr Trp Asp Thr
Thr Tyr Cys Pro 50 55 60 gac gac gtg acc tgc gca gag aat tgt gcg
ctg gat ggc gca gat tac 288Asp Asp Val Thr Cys Ala Glu Asn Cys Ala
Leu Asp Gly Ala Asp Tyr 65 70 75 gag ggc acc tac ggc gtg acc acc
tcg ggc agc tcc ctg aag ctc gat 336Glu Gly Thr Tyr Gly Val Thr Thr
Ser Gly Ser Ser Leu Lys Leu Asp 80 85 90 ttc gtc acc ggg tct aac
gtc gga tct cgt ctc tac ctg ttg gag aat 384Phe Val Thr Gly Ser Asn
Val Gly Ser Arg Leu Tyr Leu Leu Glu Asn 95 100 105 110 gat tcg acc
tat cag atc ttc aag ctt ctg aac cag gaa ttc acc ttt 432Asp Ser Thr
Tyr Gln Ile Phe Lys Leu Leu Asn Gln Glu Phe Thr Phe 115 120 125 gac
gtc gac gtt tcc aat ctt ccg tgc gga tta aac ggc gct ctg tac 480Asp
Val Asp Val Ser Asn Leu Pro Cys Gly Leu Asn Gly Ala Leu Tyr 130 135
140 ctt gtt acc atg gct gct gac ggc ggg gtg tct cag tac ccg aat aac
528Leu Val Thr Met Ala Ala Asp Gly Gly Val Ser Gln Tyr Pro Asn Asn
145 150 155 aag gcc ggc gca gcg tat gga acc ggt tat tgc gat tcc cag
tgt cca 576Lys Ala Gly Ala Ala Tyr Gly Thr Gly Tyr Cys Asp Ser Gln
Cys Pro 160 165 170 agg gac ttg aag ttt atc gat ggc cag gtatgtagag
ctgtaatcac 623Arg Asp Leu Lys Phe Ile Asp Gly Gln 175 180
ccatgttgtg aaatcactct cctactgaca tggtcgattt atag gcc aac gtt gag
679 Ala Asn Val Glu 185 ggc tgg cag ccg tct tcg aac aac gcc aat aca
ggt att ggc aac cat 727Gly Trp Gln Pro Ser Ser Asn Asn Ala Asn Thr
Gly Ile Gly Asn His 190 195 200 ggc tcc tgc tgt gcg gag atg gat atc
tgg gaa gcc aac agc atc tcc 775Gly Ser Cys Cys Ala Glu Met Asp Ile
Trp Glu Ala Asn Ser Ile Ser 205 210 215 aat gcg gtg act ccg cac cca
tgc gac aca ccc ggc cag aca atg tgc 823Asn Ala Val Thr Pro His Pro
Cys Asp Thr Pro Gly Gln Thr Met Cys 220 225 230 235 gag ggg aac gac
tgt ggt ggc acg tat tcc acc aat cgc tat gca ggc 871Glu Gly Asn Asp
Cys Gly Gly Thr Tyr Ser Thr Asn Arg Tyr Ala Gly 240 245 250 acc tgc
gat cct gac ggc tgc gac ttc aac ccc tac cgc atg ggc aac 919Thr Cys
Asp Pro Asp Gly Cys Asp Phe Asn Pro Tyr Arg Met Gly Asn 255 260 265
cat tct ttc tac ggc cct ggg gag att gtc gat act acc cag ccc ttc
967His Ser Phe Tyr Gly Pro Gly Glu Ile Val Asp Thr Thr Gln Pro Phe
270 275 280 act gtc gtg aca cag ttc ctt acc gat gat ggc acg gat act
ggc act 1015Thr Val Val Thr Gln Phe Leu Thr Asp Asp Gly Thr Asp Thr
Gly Thr 285 290 295 ctc agc gag atc aaa cgc ttc tac gtc caa aac ggg
aaa gtc att cct 1063Leu Ser Glu Ile Lys Arg Phe Tyr Val Gln Asn Gly
Lys Val Ile Pro 300 305 310 315 cag ccg aac tcc gac att gcc ggc gtg
act ggc aac tcg atc acc agc 1111Gln Pro Asn Ser Asp Ile Ala Gly Val
Thr Gly Asn Ser Ile Thr Ser 320 325 330 gag ttt tgc gat gcc cag aag
acg gct ttc ggc gac att aac aac ttt 1159Glu Phe Cys Asp Ala Gln Lys
Thr Ala Phe Gly Asp Ile Asn Asn Phe 335 340 345 gat aca cac ggc ggt
ctg gcc agt atg gga gct gcg ctg cag cag ggt 1207Asp Thr His Gly Gly
Leu Ala Ser Met Gly Ala Ala Leu Gln Gln Gly 350 355 360 atg gtt ctg
gtg atg agt ctg tgg gac g gtaggtcctt gggagacacc 1255Met Val Leu Val
Met Ser Leu Trp Asp 365 370 cggacgttct atatcaacca gaactgccag
aactgacgaa ttaaaacact tttag at 1312 Asp tac gcg gca aac atg ctg tgg
ttg gac agc att tat cca aca aat gca 1360Tyr Ala Ala Asn Met Leu Trp
Leu Asp Ser Ile Tyr Pro Thr Asn Ala 375 380 385 tct gct agc act cct
ggt gct gct cgt gga acc tgt tcg acg agc tcc 1408Ser Ala Ser Thr Pro
Gly Ala Ala Arg Gly Thr Cys Ser Thr Ser Ser 390 395 400 405 ggt gtc
cca tcg caa gtc gag tcg cag agc ccc aac gcc tac gtg acg 1456Gly Val
Pro Ser Gln Val Glu Ser Gln Ser Pro Asn Ala Tyr Val Thr 410 415 420
tac tcc aac att aaa gtt gga cca atc aac tcg acc ttc acc act tcg
1504Tyr Ser Asn Ile Lys Val Gly Pro Ile Asn Ser Thr Phe Thr Thr Ser
425 430 435 ggc tcg aac cct gga ggc gga acg acc act act aca acg act
cag ccg 1552Gly Ser Asn Pro Gly Gly Gly Thr Thr Thr Thr Thr Thr Thr
Gln Pro 440 445 450 aca aca aca act acc aca gca ggc aac cct gga ggt
aca ggt gtg gcc 1600Thr Thr Thr Thr Thr Thr Ala Gly Asn Pro Gly Gly
Thr Gly Val Ala 455 460 465 cag cac tac gga cag tgt ggc ggt atc gga
tgg aca gga cct act act 1648Gln His Tyr Gly Gln Cys Gly Gly Ile Gly
Trp Thr Gly Pro Thr Thr 470 475 480 485 tgt gca tcg cct tat acc tgt
cag aaa ttg aac gac tgg tac tcg cag 1696Cys Ala Ser Pro Tyr Thr Cys
Gln Lys Leu Asn Asp Trp Tyr Ser Gln 490 495 500 tgt ttg taa 1705Cys
Leu 57521PRTArtificial SequenceSynthetic Construct 57Met Phe Arg
Arg Ala Leu Phe Leu Ser Ser Ser Ala Phe Leu Ala Val -15 -10 -5 Lys
Ala Gln Gln Ile Gly Thr Val Ser Pro Glu Asn His Pro Pro Leu -1 1 5
10 Ala
Trp Glu Gln Cys Thr Ala Pro Gly Ser Cys Thr Thr Val Asn Gly 15 20
25 30 Ala Val Val Leu Asp Ala Asn Trp Arg Trp Val His Asn Val Gly
Gly 35 40 45 Tyr Thr Asn Cys Tyr Thr Gly Asn Thr Trp Asp Thr Thr
Tyr Cys Pro 50 55 60 Asp Asp Val Thr Cys Ala Glu Asn Cys Ala Leu
Asp Gly Ala Asp Tyr 65 70 75 Glu Gly Thr Tyr Gly Val Thr Thr Ser
Gly Ser Ser Leu Lys Leu Asp 80 85 90 Phe Val Thr Gly Ser Asn Val
Gly Ser Arg Leu Tyr Leu Leu Glu Asn 95 100 105 110 Asp Ser Thr Tyr
Gln Ile Phe Lys Leu Leu Asn Gln Glu Phe Thr Phe 115 120 125 Asp Val
Asp Val Ser Asn Leu Pro Cys Gly Leu Asn Gly Ala Leu Tyr 130 135 140
Leu Val Thr Met Ala Ala Asp Gly Gly Val Ser Gln Tyr Pro Asn Asn 145
150 155 Lys Ala Gly Ala Ala Tyr Gly Thr Gly Tyr Cys Asp Ser Gln Cys
Pro 160 165 170 Arg Asp Leu Lys Phe Ile Asp Gly Gln Ala Asn Val Glu
Gly Trp Gln 175 180 185 190 Pro Ser Ser Asn Asn Ala Asn Thr Gly Ile
Gly Asn His Gly Ser Cys 195 200 205 Cys Ala Glu Met Asp Ile Trp Glu
Ala Asn Ser Ile Ser Asn Ala Val 210 215 220 Thr Pro His Pro Cys Asp
Thr Pro Gly Gln Thr Met Cys Glu Gly Asn 225 230 235 Asp Cys Gly Gly
Thr Tyr Ser Thr Asn Arg Tyr Ala Gly Thr Cys Asp 240 245 250 Pro Asp
Gly Cys Asp Phe Asn Pro Tyr Arg Met Gly Asn His Ser Phe 255 260 265
270 Tyr Gly Pro Gly Glu Ile Val Asp Thr Thr Gln Pro Phe Thr Val Val
275 280 285 Thr Gln Phe Leu Thr Asp Asp Gly Thr Asp Thr Gly Thr Leu
Ser Glu 290 295 300 Ile Lys Arg Phe Tyr Val Gln Asn Gly Lys Val Ile
Pro Gln Pro Asn 305 310 315 Ser Asp Ile Ala Gly Val Thr Gly Asn Ser
Ile Thr Ser Glu Phe Cys 320 325 330 Asp Ala Gln Lys Thr Ala Phe Gly
Asp Ile Asn Asn Phe Asp Thr His 335 340 345 350 Gly Gly Leu Ala Ser
Met Gly Ala Ala Leu Gln Gln Gly Met Val Leu 355 360 365 Val Met Ser
Leu Trp Asp Asp Tyr Ala Ala Asn Met Leu Trp Leu Asp 370 375 380 Ser
Ile Tyr Pro Thr Asn Ala Ser Ala Ser Thr Pro Gly Ala Ala Arg 385 390
395 Gly Thr Cys Ser Thr Ser Ser Gly Val Pro Ser Gln Val Glu Ser Gln
400 405 410 Ser Pro Asn Ala Tyr Val Thr Tyr Ser Asn Ile Lys Val Gly
Pro Ile 415 420 425 430 Asn Ser Thr Phe Thr Thr Ser Gly Ser Asn Pro
Gly Gly Gly Thr Thr 435 440 445 Thr Thr Thr Thr Thr Gln Pro Thr Thr
Thr Thr Thr Thr Ala Gly Asn 450 455 460 Pro Gly Gly Thr Gly Val Ala
Gln His Tyr Gly Gln Cys Gly Gly Ile 465 470 475 Gly Trp Thr Gly Pro
Thr Thr Cys Ala Ser Pro Tyr Thr Cys Gln Lys 480 485 490 Leu Asn Asp
Trp Tyr Ser Gln Cys Leu 495 500 5849DNAArtificial SequenceSYNTHETIC
PRIMER 58atacctgtca gaaattgaac gactggtact cgcagtgttt gtaagcttc
495932DNAArtificial SequenceSYNTHETIC PRIMER 59gtcgttcaat
ttctgacagg tataaggcga tg 326023DNAArtificial SequenceSYNTHETIC
PRIMER 60cctcagccga actccgacat tgc 236123DNAArtificial
SequenceSYNTHETIC PRIMER 61gcaatgtcgg agttcggctg agg
23621705DNAArtificial
SequenceSyntheticexon(1)..(603)sig_peptide(1)..(18)CDS(1)..(603)mat_pepti-
de(55)..(1702)Intron(604)..(667)exon(668)..(1235)CDS(668)..(1235)Intron(12-
36)..(1310)exon(1311)..(1702)CDS(1311)..(1702) 62atg ttt cga cgg
gct ctt ttc ctg tcc tct tcc gcc ttc ctt gct gtc 48Met Phe Arg Arg
Ala Leu Phe Leu Ser Ser Ser Ala Phe Leu Ala Val -15 -10 -5 aaa gcc
cag cag atc ggc acg gtc agt ccg gag aac cat ccg ccc ctg 96Lys Ala
Gln Gln Ile Gly Thr Val Ser Pro Glu Asn His Pro Pro Leu -1 1 5 10
gca tgg gag cag tgc act gcc cct ggg agt tgc acg act gtg aat ggt
144Ala Trp Glu Gln Cys Thr Ala Pro Gly Ser Cys Thr Thr Val Asn Gly
15 20 25 30 gcg gtc gtc ctt gat gcg aac tgg cgt tgg gtc cac aat gtt
ggg gga 192Ala Val Val Leu Asp Ala Asn Trp Arg Trp Val His Asn Val
Gly Gly 35 40 45 tac acc aac tgc tac act ggc aat acc tgg gac acc
acg tac tgc cct 240Tyr Thr Asn Cys Tyr Thr Gly Asn Thr Trp Asp Thr
Thr Tyr Cys Pro 50 55 60 gac gac gtg acc tgc gca gag aat tgt gcg
ctg gat ggc gca gat tac 288Asp Asp Val Thr Cys Ala Glu Asn Cys Ala
Leu Asp Gly Ala Asp Tyr 65 70 75 gag ggc acc tac ggc gtg acc acc
tcg ggc agc tcc ctg aag ctc gat 336Glu Gly Thr Tyr Gly Val Thr Thr
Ser Gly Ser Ser Leu Lys Leu Asp 80 85 90 ttc gtc acc ggg tct aac
gtc gga tct cgt ctc tac ctg ttg gag aat 384Phe Val Thr Gly Ser Asn
Val Gly Ser Arg Leu Tyr Leu Leu Glu Asn 95 100 105 110 gat tcg acc
tat cag atc ttc aag ctt ctg aac cag gaa ttc acc ttt 432Asp Ser Thr
Tyr Gln Ile Phe Lys Leu Leu Asn Gln Glu Phe Thr Phe 115 120 125 gac
gtc gac gtt tcc aat ctt ccg tgc gga tta aac ggc gct ctg tac 480Asp
Val Asp Val Ser Asn Leu Pro Cys Gly Leu Asn Gly Ala Leu Tyr 130 135
140 ctt gtt acc atg gct gct gac ggc ggg gtg tct cag tac ccg aat aac
528Leu Val Thr Met Ala Ala Asp Gly Gly Val Ser Gln Tyr Pro Asn Asn
145 150 155 aag gcc ggc gca gcg tat gga acc ggt tat tgc gat tcc cag
tgt cca 576Lys Ala Gly Ala Ala Tyr Gly Thr Gly Tyr Cys Asp Ser Gln
Cys Pro 160 165 170 agg gac ttg aag ttt atc gat ggc cag gtatgtagag
ctgtaatcac 623Arg Asp Leu Lys Phe Ile Asp Gly Gln 175 180
ccatgttgtg aaatcactct cctactgaca tggtcgattt atag gcc aac gtt gag
679 Ala Asn Val Glu 185 ggc tgg cag ccg tct tcg aac aac gcc aat aca
ggt att ggc aac cat 727Gly Trp Gln Pro Ser Ser Asn Asn Ala Asn Thr
Gly Ile Gly Asn His 190 195 200 ggc tcc tgc tgt gcg gag atg gat atc
tgg gaa gcc aac agc atc tcc 775Gly Ser Cys Cys Ala Glu Met Asp Ile
Trp Glu Ala Asn Ser Ile Ser 205 210 215 aat gcg gtg act ccg cac cca
tgc gac aca ccc ggc cag aca atg tgc 823Asn Ala Val Thr Pro His Pro
Cys Asp Thr Pro Gly Gln Thr Met Cys 220 225 230 235 gag ggg aac gac
tgt ggt ggc acg tat tcc acc aat cgc tat gca ggc 871Glu Gly Asn Asp
Cys Gly Gly Thr Tyr Ser Thr Asn Arg Tyr Ala Gly 240 245 250 acc tgc
gat cct gac ggc tgc gac ttc aac ccc tac cgc atg ggc aac 919Thr Cys
Asp Pro Asp Gly Cys Asp Phe Asn Pro Tyr Arg Met Gly Asn 255 260 265
cat tct ttc tac ggc cct ggg gag att gtc gat act acc cag ccc ttc
967His Ser Phe Tyr Gly Pro Gly Glu Ile Val Asp Thr Thr Gln Pro Phe
270 275 280 acg gtc gtg aca cag ttc ctt acc gat gat ggc acg gat act
ggc act 1015Thr Val Val Thr Gln Phe Leu Thr Asp Asp Gly Thr Asp Thr
Gly Thr 285 290 295 ctc agc gag atc aaa cgc ttc tac gtc caa aac ggg
aaa gtc att cct 1063Leu Ser Glu Ile Lys Arg Phe Tyr Val Gln Asn Gly
Lys Val Ile Pro 300 305 310 315 cag ccg aac tcc gac att gcc ggc gtg
act ggc aac tcg atc acc agc 1111Gln Pro Asn Ser Asp Ile Ala Gly Val
Thr Gly Asn Ser Ile Thr Ser 320 325 330 gag ttt tgc gat gcc cag aag
acg gct ttc ggc gac att aac aac ttt 1159Glu Phe Cys Asp Ala Gln Lys
Thr Ala Phe Gly Asp Ile Asn Asn Phe 335 340 345 gat aca cac ggc ggt
ctg gcc agt atg gga gct gcg ctg cag cag ggt 1207Asp Thr His Gly Gly
Leu Ala Ser Met Gly Ala Ala Leu Gln Gln Gly 350 355 360 atg gtt ctg
gtg atg agt ctg tgg gac g gtaggtcctt gggagacacc 1255Met Val Leu Val
Met Ser Leu Trp Asp 365 370 cggacgttct atatcaacca gaactgccag
aactgacgaa ttaaaacact tttag at 1312 Asp tac gcg gca aac atg ctg tgg
ttg gac agc att tat cca aca gac gca 1360Tyr Ala Ala Asn Met Leu Trp
Leu Asp Ser Ile Tyr Pro Thr Asp Ala 375 380 385 gac cct agc act cct
ggt gct gct cgt gga acc tgt tcg acg agc tcc 1408Asp Pro Ser Thr Pro
Gly Ala Ala Arg Gly Thr Cys Ser Thr Ser Ser 390 395 400 405 ggt gtc
cca tcg caa gtc gag tcg cag agc ccc aac gcc tac gtg acg 1456Gly Val
Pro Ser Gln Val Glu Ser Gln Ser Pro Asn Ala Tyr Val Thr 410 415 420
tac tcc aac att aaa gtt gga cca atc aac tcg acc ttc acc act tcg
1504Tyr Ser Asn Ile Lys Val Gly Pro Ile Asn Ser Thr Phe Thr Thr Ser
425 430 435 ggc tcg aac cct gga ggc gga acg acc act act aca acg act
cag ccg 1552Gly Ser Asn Pro Gly Gly Gly Thr Thr Thr Thr Thr Thr Thr
Gln Pro 440 445 450 aca aca aca act acc aca gca ggc aac cct gga ggt
aca ggt gtg gcc 1600Thr Thr Thr Thr Thr Thr Ala Gly Asn Pro Gly Gly
Thr Gly Val Ala 455 460 465 cag cac tac gga cag tgt ggc ggt atc gga
tgg aca gga cct act act 1648Gln His Tyr Gly Gln Cys Gly Gly Ile Gly
Trp Thr Gly Pro Thr Thr 470 475 480 485 tgt gca tcg cct tat acc tgt
cag aaa ttg aac gac tgg tac tcg cag 1696Cys Ala Ser Pro Tyr Thr Cys
Gln Lys Leu Asn Asp Trp Tyr Ser Gln 490 495 500 tgt ttg taa 1705Cys
Leu 63521PRTArtificial SequenceSynthetic Construct 63Met Phe Arg
Arg Ala Leu Phe Leu Ser Ser Ser Ala Phe Leu Ala Val -15 -10 -5 Lys
Ala Gln Gln Ile Gly Thr Val Ser Pro Glu Asn His Pro Pro Leu -1 1 5
10 Ala Trp Glu Gln Cys Thr Ala Pro Gly Ser Cys Thr Thr Val Asn Gly
15 20 25 30 Ala Val Val Leu Asp Ala Asn Trp Arg Trp Val His Asn Val
Gly Gly 35 40 45 Tyr Thr Asn Cys Tyr Thr Gly Asn Thr Trp Asp Thr
Thr Tyr Cys Pro 50 55 60 Asp Asp Val Thr Cys Ala Glu Asn Cys Ala
Leu Asp Gly Ala Asp Tyr 65 70 75 Glu Gly Thr Tyr Gly Val Thr Thr
Ser Gly Ser Ser Leu Lys Leu Asp 80 85 90 Phe Val Thr Gly Ser Asn
Val Gly Ser Arg Leu Tyr Leu Leu Glu Asn 95 100 105 110 Asp Ser Thr
Tyr Gln Ile Phe Lys Leu Leu Asn Gln Glu Phe Thr Phe 115 120 125 Asp
Val Asp Val Ser Asn Leu Pro Cys Gly Leu Asn Gly Ala Leu Tyr 130 135
140 Leu Val Thr Met Ala Ala Asp Gly Gly Val Ser Gln Tyr Pro Asn Asn
145 150 155 Lys Ala Gly Ala Ala Tyr Gly Thr Gly Tyr Cys Asp Ser Gln
Cys Pro 160 165 170 Arg Asp Leu Lys Phe Ile Asp Gly Gln Ala Asn Val
Glu Gly Trp Gln 175 180 185 190 Pro Ser Ser Asn Asn Ala Asn Thr Gly
Ile Gly Asn His Gly Ser Cys 195 200 205 Cys Ala Glu Met Asp Ile Trp
Glu Ala Asn Ser Ile Ser Asn Ala Val 210 215 220 Thr Pro His Pro Cys
Asp Thr Pro Gly Gln Thr Met Cys Glu Gly Asn 225 230 235 Asp Cys Gly
Gly Thr Tyr Ser Thr Asn Arg Tyr Ala Gly Thr Cys Asp 240 245 250 Pro
Asp Gly Cys Asp Phe Asn Pro Tyr Arg Met Gly Asn His Ser Phe 255 260
265 270 Tyr Gly Pro Gly Glu Ile Val Asp Thr Thr Gln Pro Phe Thr Val
Val 275 280 285 Thr Gln Phe Leu Thr Asp Asp Gly Thr Asp Thr Gly Thr
Leu Ser Glu 290 295 300 Ile Lys Arg Phe Tyr Val Gln Asn Gly Lys Val
Ile Pro Gln Pro Asn 305 310 315 Ser Asp Ile Ala Gly Val Thr Gly Asn
Ser Ile Thr Ser Glu Phe Cys 320 325 330 Asp Ala Gln Lys Thr Ala Phe
Gly Asp Ile Asn Asn Phe Asp Thr His 335 340 345 350 Gly Gly Leu Ala
Ser Met Gly Ala Ala Leu Gln Gln Gly Met Val Leu 355 360 365 Val Met
Ser Leu Trp Asp Asp Tyr Ala Ala Asn Met Leu Trp Leu Asp 370 375 380
Ser Ile Tyr Pro Thr Asp Ala Asp Pro Ser Thr Pro Gly Ala Ala Arg 385
390 395 Gly Thr Cys Ser Thr Ser Ser Gly Val Pro Ser Gln Val Glu Ser
Gln 400 405 410 Ser Pro Asn Ala Tyr Val Thr Tyr Ser Asn Ile Lys Val
Gly Pro Ile 415 420 425 430 Asn Ser Thr Phe Thr Thr Ser Gly Ser Asn
Pro Gly Gly Gly Thr Thr 435 440 445 Thr Thr Thr Thr Thr Gln Pro Thr
Thr Thr Thr Thr Thr Ala Gly Asn 450 455 460 Pro Gly Gly Thr Gly Val
Ala Gln His Tyr Gly Gln Cys Gly Gly Ile 465 470 475 Gly Trp Thr Gly
Pro Thr Thr Cys Ala Ser Pro Tyr Thr Cys Gln Lys 480 485 490 Leu Asn
Asp Trp Tyr Ser Gln Cys Leu 495 500 6455DNAArtificial
SequenceSYNTHETIC PRIMER 64ggttggacag catttatcca acagacgcag
accctagcac tcctggtgct gctcg 556535DNAArtificial SequenceSYNTHETIC
PRIMER 65tgttggataa atgctgtcca accacagcat gtttg
35661599DNAAspergillus
fumigatussig_peptide(1)..(26)CDS(1)..(1596)mat_peptide(79)..(1596)
66atg ttg gcc tcc acg ttc tcc tat cgc atg tac aaa aca gcg ctc atc
48Met Leu Ala Ser Thr Phe Ser Tyr Arg Met Tyr Lys Thr Ala Leu Ile
-25 -20 -15 ttg gca gcc ctc ttg ggc tcg gga cag gca cag cag gtc tgt
acc tcg 96Leu Ala Ala Leu Leu Gly Ser Gly Gln Ala Gln Gln Val Cys
Thr Ser -10 -5 -1 1 5 cag gcc gag gtc cat cct tcc atg acg tgg cag
tcg tgt aca gcg ggt 144Gln Ala Glu Val His Pro Ser Met Thr Trp Gln
Ser Cys Thr Ala Gly 10 15 20 ggt tcg tgt acc aca aac aac ggt aaa
gtc gtg atc gat gca aac tgg 192Gly Ser Cys Thr Thr Asn Asn Gly Lys
Val Val Ile Asp Ala Asn Trp 25 30 35 agg tgg gtg cac aag gtc ggc
gac tac acc aac tgt tac aca ggc aac 240Arg Trp Val His Lys Val Gly
Asp Tyr Thr Asn Cys Tyr Thr Gly Asn 40 45 50 aca tgg gat aca acc
atc tgt ccc gac gat gcc act tgt gca tcc aac 288Thr Trp Asp Thr Thr
Ile Cys Pro Asp Asp Ala Thr Cys Ala Ser Asn 55 60 65 70 tgt tgt ctc
gag ggt gcc aac tat gag tcg acg tac gga gtg acc gcc 336Cys Cys Leu
Glu Gly Ala Asn Tyr Glu Ser Thr Tyr Gly Val Thr Ala 75 80 85 tcc
gga aac tcg ctc agg
ctc aac ttc gtc aca act tcc cag cag aag 384Ser Gly Asn Ser Leu Arg
Leu Asn Phe Val Thr Thr Ser Gln Gln Lys 90 95 100 aac atc ggc tcg
cgg ttg tat atg atg aaa gac gat tcc act tac gag 432Asn Ile Gly Ser
Arg Leu Tyr Met Met Lys Asp Asp Ser Thr Tyr Glu 105 110 115 atg ttc
aag ctc ctc aac cag gaa ttc act ttc gat gtc gac gtc tcc 480Met Phe
Lys Leu Leu Asn Gln Glu Phe Thr Phe Asp Val Asp Val Ser 120 125 130
aac ctc cct tgt ggc ttg aac gga gcg ctc tac ttc gtc gcc atg gat
528Asn Leu Pro Cys Gly Leu Asn Gly Ala Leu Tyr Phe Val Ala Met Asp
135 140 145 150 gcg gat gga ggc atg tcc aag tat cct acc aac aaa gca
gga gcc aag 576Ala Asp Gly Gly Met Ser Lys Tyr Pro Thr Asn Lys Ala
Gly Ala Lys 155 160 165 tat ggt aca ggt tac tgt gat tcc cag tgt ccc
agg gat ctc aag ttc 624Tyr Gly Thr Gly Tyr Cys Asp Ser Gln Cys Pro
Arg Asp Leu Lys Phe 170 175 180 atc aac ggt cag gcc aac gtc gag ggt
tgg cag cct tcg tcg aac gat 672Ile Asn Gly Gln Ala Asn Val Glu Gly
Trp Gln Pro Ser Ser Asn Asp 185 190 195 gcc aac gca ggt acc ggc aac
cac ggt tcc tgt tgt gcc gaa atg gac 720Ala Asn Ala Gly Thr Gly Asn
His Gly Ser Cys Cys Ala Glu Met Asp 200 205 210 att tgg gaa gcg aac
tcg atc tcg acg gcg ttc act cct cac ccg tgt 768Ile Trp Glu Ala Asn
Ser Ile Ser Thr Ala Phe Thr Pro His Pro Cys 215 220 225 230 gat aca
ccc gga cag gtg atg tgt aca ggc gac gcc tgt ggc gga acc 816Asp Thr
Pro Gly Gln Val Met Cys Thr Gly Asp Ala Cys Gly Gly Thr 235 240 245
tac tcg tcg gat cga tat ggc ggt acg tgt gac ccc gac ggc tgt gac
864Tyr Ser Ser Asp Arg Tyr Gly Gly Thr Cys Asp Pro Asp Gly Cys Asp
250 255 260 ttc aac tcc ttc agg cag ggc aac aaa aca ttc tat gga cct
ggc atg 912Phe Asn Ser Phe Arg Gln Gly Asn Lys Thr Phe Tyr Gly Pro
Gly Met 265 270 275 acg gtg gat aca aag tcg aaa ttc aca gtc gtc act
cag ttc atc acc 960Thr Val Asp Thr Lys Ser Lys Phe Thr Val Val Thr
Gln Phe Ile Thr 280 285 290 gac gat ggt acg tcc tcg ggt acc ttg aag
gag atc aaa agg ttc tat 1008Asp Asp Gly Thr Ser Ser Gly Thr Leu Lys
Glu Ile Lys Arg Phe Tyr 295 300 305 310 gtc cag aac gga aag gtc atc
ccg aac tcg gag tcc acg tgg aca gga 1056Val Gln Asn Gly Lys Val Ile
Pro Asn Ser Glu Ser Thr Trp Thr Gly 315 320 325 gtg tcg ggt aac tcc
atc act acg gag tat tgt aca gcc cag aag tcg 1104Val Ser Gly Asn Ser
Ile Thr Thr Glu Tyr Cys Thr Ala Gln Lys Ser 330 335 340 ctc ttc cag
gat cag aac gtc ttc gag aaa cat gga ggc ttg gaa gga 1152Leu Phe Gln
Asp Gln Asn Val Phe Glu Lys His Gly Gly Leu Glu Gly 345 350 355 atg
ggt gcc gca ttg gcc cag ggt atg gtc ctc gtc atg tcc ttg tgg 1200Met
Gly Ala Ala Leu Ala Gln Gly Met Val Leu Val Met Ser Leu Trp 360 365
370 gac gac cac tcg gcc aac atg ctc tgg ttg gat tcc aac tac ccc acc
1248Asp Asp His Ser Ala Asn Met Leu Trp Leu Asp Ser Asn Tyr Pro Thr
375 380 385 390 act gcc tcg tcc acg aca ccg ggt gtc gca cgc gga act
tgt gat atc 1296Thr Ala Ser Ser Thr Thr Pro Gly Val Ala Arg Gly Thr
Cys Asp Ile 395 400 405 tcc tcg gga gtg cct gca gac gtc gag gcg aac
cat ccc gac gcc tac 1344Ser Ser Gly Val Pro Ala Asp Val Glu Ala Asn
His Pro Asp Ala Tyr 410 415 420 gtg gtc tac tcg aac att aag gtg gga
ccc atc ggt tcg aca ttc aac 1392Val Val Tyr Ser Asn Ile Lys Val Gly
Pro Ile Gly Ser Thr Phe Asn 425 430 435 tcc gga ggc tcg aac cct gga
ggc gga acg acc act act aca acg act 1440Ser Gly Gly Ser Asn Pro Gly
Gly Gly Thr Thr Thr Thr Thr Thr Thr 440 445 450 cag ccg aca aca aca
act acc aca gca ggc aac cct gga ggt aca ggt 1488Gln Pro Thr Thr Thr
Thr Thr Thr Ala Gly Asn Pro Gly Gly Thr Gly 455 460 465 470 gtg gcc
cag cac tac gga cag tgt ggc ggt atc gga tgg aca gga cct 1536Val Ala
Gln His Tyr Gly Gln Cys Gly Gly Ile Gly Trp Thr Gly Pro 475 480 485
act act tgt gca tcg cct tat acc tgt cag aaa ttg aac gac tac tac
1584Thr Thr Cys Ala Ser Pro Tyr Thr Cys Gln Lys Leu Asn Asp Tyr Tyr
490 495 500 tcg cag tgt ttg taa 1599Ser Gln Cys Leu 505
67532PRTAspergillus fumigatus 67Met Leu Ala Ser Thr Phe Ser Tyr Arg
Met Tyr Lys Thr Ala Leu Ile -25 -20 -15 Leu Ala Ala Leu Leu Gly Ser
Gly Gln Ala Gln Gln Val Cys Thr Ser -10 -5 -1 1 5 Gln Ala Glu Val
His Pro Ser Met Thr Trp Gln Ser Cys Thr Ala Gly 10 15 20 Gly Ser
Cys Thr Thr Asn Asn Gly Lys Val Val Ile Asp Ala Asn Trp 25 30 35
Arg Trp Val His Lys Val Gly Asp Tyr Thr Asn Cys Tyr Thr Gly Asn 40
45 50 Thr Trp Asp Thr Thr Ile Cys Pro Asp Asp Ala Thr Cys Ala Ser
Asn 55 60 65 70 Cys Cys Leu Glu Gly Ala Asn Tyr Glu Ser Thr Tyr Gly
Val Thr Ala 75 80 85 Ser Gly Asn Ser Leu Arg Leu Asn Phe Val Thr
Thr Ser Gln Gln Lys 90 95 100 Asn Ile Gly Ser Arg Leu Tyr Met Met
Lys Asp Asp Ser Thr Tyr Glu 105 110 115 Met Phe Lys Leu Leu Asn Gln
Glu Phe Thr Phe Asp Val Asp Val Ser 120 125 130 Asn Leu Pro Cys Gly
Leu Asn Gly Ala Leu Tyr Phe Val Ala Met Asp 135 140 145 150 Ala Asp
Gly Gly Met Ser Lys Tyr Pro Thr Asn Lys Ala Gly Ala Lys 155 160 165
Tyr Gly Thr Gly Tyr Cys Asp Ser Gln Cys Pro Arg Asp Leu Lys Phe 170
175 180 Ile Asn Gly Gln Ala Asn Val Glu Gly Trp Gln Pro Ser Ser Asn
Asp 185 190 195 Ala Asn Ala Gly Thr Gly Asn His Gly Ser Cys Cys Ala
Glu Met Asp 200 205 210 Ile Trp Glu Ala Asn Ser Ile Ser Thr Ala Phe
Thr Pro His Pro Cys 215 220 225 230 Asp Thr Pro Gly Gln Val Met Cys
Thr Gly Asp Ala Cys Gly Gly Thr 235 240 245 Tyr Ser Ser Asp Arg Tyr
Gly Gly Thr Cys Asp Pro Asp Gly Cys Asp 250 255 260 Phe Asn Ser Phe
Arg Gln Gly Asn Lys Thr Phe Tyr Gly Pro Gly Met 265 270 275 Thr Val
Asp Thr Lys Ser Lys Phe Thr Val Val Thr Gln Phe Ile Thr 280 285 290
Asp Asp Gly Thr Ser Ser Gly Thr Leu Lys Glu Ile Lys Arg Phe Tyr 295
300 305 310 Val Gln Asn Gly Lys Val Ile Pro Asn Ser Glu Ser Thr Trp
Thr Gly 315 320 325 Val Ser Gly Asn Ser Ile Thr Thr Glu Tyr Cys Thr
Ala Gln Lys Ser 330 335 340 Leu Phe Gln Asp Gln Asn Val Phe Glu Lys
His Gly Gly Leu Glu Gly 345 350 355 Met Gly Ala Ala Leu Ala Gln Gly
Met Val Leu Val Met Ser Leu Trp 360 365 370 Asp Asp His Ser Ala Asn
Met Leu Trp Leu Asp Ser Asn Tyr Pro Thr 375 380 385 390 Thr Ala Ser
Ser Thr Thr Pro Gly Val Ala Arg Gly Thr Cys Asp Ile 395 400 405 Ser
Ser Gly Val Pro Ala Asp Val Glu Ala Asn His Pro Asp Ala Tyr 410 415
420 Val Val Tyr Ser Asn Ile Lys Val Gly Pro Ile Gly Ser Thr Phe Asn
425 430 435 Ser Gly Gly Ser Asn Pro Gly Gly Gly Thr Thr Thr Thr Thr
Thr Thr 440 445 450 Gln Pro Thr Thr Thr Thr Thr Thr Ala Gly Asn Pro
Gly Gly Thr Gly 455 460 465 470 Val Ala Gln His Tyr Gly Gln Cys Gly
Gly Ile Gly Trp Thr Gly Pro 475 480 485 Thr Thr Cys Ala Ser Pro Tyr
Thr Cys Gln Lys Leu Asn Asp Tyr Tyr 490 495 500 Ser Gln Cys Leu 505
681599DNAAspergillus
fumigatussig_peptide(1)..(26)CDS(1)..(1596)mat_peptide(79)..(1596)
68atg ttg gcc tcc acg ttc tcc tat cgc atg tac aaa aca gcg ctc atc
48Met Leu Ala Ser Thr Phe Ser Tyr Arg Met Tyr Lys Thr Ala Leu Ile
-25 -20 -15 ttg gca gcc ctc ttg ggc tcg gga cag gca cag cag gtc gga
acc tcg 96Leu Ala Ala Leu Leu Gly Ser Gly Gln Ala Gln Gln Val Gly
Thr Ser -10 -5 -1 1 5 cag gcc gag gtc cat cct tcc atg acg tgg cag
tcg tgt aca gcg ggt 144Gln Ala Glu Val His Pro Ser Met Thr Trp Gln
Ser Cys Thr Ala Gly 10 15 20 ggt tcg tgt acc aca aac aac ggt aaa
gtc gtg atc gat gca aac tgg 192Gly Ser Cys Thr Thr Asn Asn Gly Lys
Val Val Ile Asp Ala Asn Trp 25 30 35 agg tgg gtg cac aag act ggc
gac tac acc aac tgt tac aca ggc aac 240Arg Trp Val His Lys Thr Gly
Asp Tyr Thr Asn Cys Tyr Thr Gly Asn 40 45 50 aca tgg gat aca acc
atc tgt ccc gac gat gcc act tgt gca tcc aac 288Thr Trp Asp Thr Thr
Ile Cys Pro Asp Asp Ala Thr Cys Ala Ser Asn 55 60 65 70 tgt gca ctc
gag ggt gcc aac tat gag tcg acg tac gga gtg acc gcc 336Cys Ala Leu
Glu Gly Ala Asn Tyr Glu Ser Thr Tyr Gly Val Thr Ala 75 80 85 tcc
gga aac tcg ctc agg ctc aac ttc gtc aca act tcc cag cag aag 384Ser
Gly Asn Ser Leu Arg Leu Asn Phe Val Thr Thr Ser Gln Gln Lys 90 95
100 aac atc ggc tcg cgg ttg tat atg atg aaa gac gat tcc act tac gag
432Asn Ile Gly Ser Arg Leu Tyr Met Met Lys Asp Asp Ser Thr Tyr Glu
105 110 115 atg ttc aag ctc ctc aac cag gaa ttc act ttc gat gtc gac
gtc tcc 480Met Phe Lys Leu Leu Asn Gln Glu Phe Thr Phe Asp Val Asp
Val Ser 120 125 130 aac ctc cct tgt ggc ttg aac gga gcg ctc tac ttc
gtc gcc atg gat 528Asn Leu Pro Cys Gly Leu Asn Gly Ala Leu Tyr Phe
Val Ala Met Asp 135 140 145 150 gcg gat gga ggc atg tcc aag tat cct
acc aac aaa gca gga gcc aag 576Ala Asp Gly Gly Met Ser Lys Tyr Pro
Thr Asn Lys Ala Gly Ala Lys 155 160 165 tat ggt aca ggt tac tgt gat
tcc cag tgt ccc agg gat ctc aag ttc 624Tyr Gly Thr Gly Tyr Cys Asp
Ser Gln Cys Pro Arg Asp Leu Lys Phe 170 175 180 atc aac ggt cag gcc
aac gtc gag ggt tgg cag cct tcg tcg aac gat 672Ile Asn Gly Gln Ala
Asn Val Glu Gly Trp Gln Pro Ser Ser Asn Asp 185 190 195 gcc aac gca
ggt acc ggc aac cac ggt tcc tgt tgt gcc gaa atg gac 720Ala Asn Ala
Gly Thr Gly Asn His Gly Ser Cys Cys Ala Glu Met Asp 200 205 210 att
tgg gaa gcg aac tcg atc tcg acg gcg ttc act cct cac ccg tgt 768Ile
Trp Glu Ala Asn Ser Ile Ser Thr Ala Phe Thr Pro His Pro Cys 215 220
225 230 gat aca ccc gga cag gtg atg tgt aca ggc gac gcc tgt ggc gga
acc 816Asp Thr Pro Gly Gln Val Met Cys Thr Gly Asp Ala Cys Gly Gly
Thr 235 240 245 tac tcg tcg gat cga tat ggc ggt acg tgt gac ccc gac
ggc tgt gac 864Tyr Ser Ser Asp Arg Tyr Gly Gly Thr Cys Asp Pro Asp
Gly Cys Asp 250 255 260 ttc aac cct ttc agg cag ggc aac aaa aca ttc
tat gga cct ggc atg 912Phe Asn Pro Phe Arg Gln Gly Asn Lys Thr Phe
Tyr Gly Pro Gly Met 265 270 275 acg gtg gat aca aag tcg aaa ttc aca
gtc gtc act cag ttc atc acc 960Thr Val Asp Thr Lys Ser Lys Phe Thr
Val Val Thr Gln Phe Ile Thr 280 285 290 gac gat ggt acg tcc tcg ggt
acc ttg aag gag atc aaa agg ttc tat 1008Asp Asp Gly Thr Ser Ser Gly
Thr Leu Lys Glu Ile Lys Arg Phe Tyr 295 300 305 310 gtc cag aac gga
aag gtc atc ccg aac tcg gag tcc acg tgg aca gga 1056Val Gln Asn Gly
Lys Val Ile Pro Asn Ser Glu Ser Thr Trp Thr Gly 315 320 325 gtg tcg
ggt aac tcc atc act acg gag tat tgt aca gcc cag aag tcg 1104Val Ser
Gly Asn Ser Ile Thr Thr Glu Tyr Cys Thr Ala Gln Lys Ser 330 335 340
ctc ttc cag gat cag aac gtc ttc gag aaa cat gga ggc ttg gaa gga
1152Leu Phe Gln Asp Gln Asn Val Phe Glu Lys His Gly Gly Leu Glu Gly
345 350 355 atg ggt gcc gca ttg gcc cag ggt atg gtc ctc gtc atg tcc
ttg tgg 1200Met Gly Ala Ala Leu Ala Gln Gly Met Val Leu Val Met Ser
Leu Trp 360 365 370 gac gac cac tcg gcc aac atg ctc tgg ttg gat tcc
aac tac ccc acc 1248Asp Asp His Ser Ala Asn Met Leu Trp Leu Asp Ser
Asn Tyr Pro Thr 375 380 385 390 act gcc tcg tcc acg aca ccg ggt gtc
gca cgc gga act tgt gat atc 1296Thr Ala Ser Ser Thr Thr Pro Gly Val
Ala Arg Gly Thr Cys Asp Ile 395 400 405 tcc tcg gga gtg cct gca gac
gtc gag gcg aac cat ccc gac gcc tac 1344Ser Ser Gly Val Pro Ala Asp
Val Glu Ala Asn His Pro Asp Ala Tyr 410 415 420 gtg gtc tac tcg aac
att aag gtg gga ccc atc ggt tcg aca ttc aac 1392Val Val Tyr Ser Asn
Ile Lys Val Gly Pro Ile Gly Ser Thr Phe Asn 425 430 435 tcc gga ggc
tcg aac cct gga ggc gga acg acc act act aca acg act 1440Ser Gly Gly
Ser Asn Pro Gly Gly Gly Thr Thr Thr Thr Thr Thr Thr 440 445 450 cag
ccg aca aca aca act acc aca gca ggc aac cct gga ggt aca ggt 1488Gln
Pro Thr Thr Thr Thr Thr Thr Ala Gly Asn Pro Gly Gly Thr Gly 455 460
465 470 gtg gcc cag cac tac gga cag tgt ggc ggt atc gga tgg aca gga
cct 1536Val Ala Gln His Tyr Gly Gln Cys Gly Gly Ile Gly Trp Thr Gly
Pro 475 480 485 act act tgt gca tcg cct tat acc tgt cag aaa ttg aac
gac tac tac 1584Thr Thr Cys Ala Ser Pro Tyr Thr Cys Gln Lys Leu Asn
Asp Tyr Tyr 490 495 500 tcg cag tgt ttg taa 1599Ser Gln Cys Leu 505
69532PRTAspergillus fumigatus 69Met Leu Ala Ser Thr Phe Ser Tyr Arg
Met Tyr Lys Thr Ala Leu Ile -25 -20 -15 Leu Ala Ala Leu Leu Gly Ser
Gly Gln Ala Gln Gln Val Gly Thr Ser -10 -5 -1 1 5 Gln Ala Glu Val
His Pro Ser Met Thr Trp Gln Ser Cys Thr Ala Gly 10 15 20 Gly Ser
Cys Thr Thr Asn Asn Gly Lys Val Val Ile Asp Ala Asn Trp 25 30 35
Arg Trp Val His Lys Thr Gly Asp Tyr Thr Asn Cys Tyr Thr Gly Asn 40
45 50 Thr Trp Asp Thr Thr Ile Cys Pro Asp Asp Ala Thr Cys
Ala Ser Asn 55 60 65 70 Cys Ala Leu Glu Gly Ala Asn Tyr Glu Ser Thr
Tyr Gly Val Thr Ala 75 80 85 Ser Gly Asn Ser Leu Arg Leu Asn Phe
Val Thr Thr Ser Gln Gln Lys 90 95 100 Asn Ile Gly Ser Arg Leu Tyr
Met Met Lys Asp Asp Ser Thr Tyr Glu 105 110 115 Met Phe Lys Leu Leu
Asn Gln Glu Phe Thr Phe Asp Val Asp Val Ser 120 125 130 Asn Leu Pro
Cys Gly Leu Asn Gly Ala Leu Tyr Phe Val Ala Met Asp 135 140 145 150
Ala Asp Gly Gly Met Ser Lys Tyr Pro Thr Asn Lys Ala Gly Ala Lys 155
160 165 Tyr Gly Thr Gly Tyr Cys Asp Ser Gln Cys Pro Arg Asp Leu Lys
Phe 170 175 180 Ile Asn Gly Gln Ala Asn Val Glu Gly Trp Gln Pro Ser
Ser Asn Asp 185 190 195 Ala Asn Ala Gly Thr Gly Asn His Gly Ser Cys
Cys Ala Glu Met Asp 200 205 210 Ile Trp Glu Ala Asn Ser Ile Ser Thr
Ala Phe Thr Pro His Pro Cys 215 220 225 230 Asp Thr Pro Gly Gln Val
Met Cys Thr Gly Asp Ala Cys Gly Gly Thr 235 240 245 Tyr Ser Ser Asp
Arg Tyr Gly Gly Thr Cys Asp Pro Asp Gly Cys Asp 250 255 260 Phe Asn
Pro Phe Arg Gln Gly Asn Lys Thr Phe Tyr Gly Pro Gly Met 265 270 275
Thr Val Asp Thr Lys Ser Lys Phe Thr Val Val Thr Gln Phe Ile Thr 280
285 290 Asp Asp Gly Thr Ser Ser Gly Thr Leu Lys Glu Ile Lys Arg Phe
Tyr 295 300 305 310 Val Gln Asn Gly Lys Val Ile Pro Asn Ser Glu Ser
Thr Trp Thr Gly 315 320 325 Val Ser Gly Asn Ser Ile Thr Thr Glu Tyr
Cys Thr Ala Gln Lys Ser 330 335 340 Leu Phe Gln Asp Gln Asn Val Phe
Glu Lys His Gly Gly Leu Glu Gly 345 350 355 Met Gly Ala Ala Leu Ala
Gln Gly Met Val Leu Val Met Ser Leu Trp 360 365 370 Asp Asp His Ser
Ala Asn Met Leu Trp Leu Asp Ser Asn Tyr Pro Thr 375 380 385 390 Thr
Ala Ser Ser Thr Thr Pro Gly Val Ala Arg Gly Thr Cys Asp Ile 395 400
405 Ser Ser Gly Val Pro Ala Asp Val Glu Ala Asn His Pro Asp Ala Tyr
410 415 420 Val Val Tyr Ser Asn Ile Lys Val Gly Pro Ile Gly Ser Thr
Phe Asn 425 430 435 Ser Gly Gly Ser Asn Pro Gly Gly Gly Thr Thr Thr
Thr Thr Thr Thr 440 445 450 Gln Pro Thr Thr Thr Thr Thr Thr Ala Gly
Asn Pro Gly Gly Thr Gly 455 460 465 470 Val Ala Gln His Tyr Gly Gln
Cys Gly Gly Ile Gly Trp Thr Gly Pro 475 480 485 Thr Thr Cys Ala Ser
Pro Tyr Thr Cys Gln Lys Leu Asn Asp Tyr Tyr 490 495 500 Ser Gln Cys
Leu 505 701599DNAAspergillus
fumigatussig_peptide(1)..(26)CDS(1)..(1596)mat_peptide(79)..(1596)
70atg ttg gcc tcc acg ttc tcc tat cgc atg tac aaa aca gcg ctc atc
48Met Leu Ala Ser Thr Phe Ser Tyr Arg Met Tyr Lys Thr Ala Leu Ile
-25 -20 -15 ttg gca gcc ctc ttg ggc tcg gga cag gca cag cag gtc gga
acc tcg 96Leu Ala Ala Leu Leu Gly Ser Gly Gln Ala Gln Gln Val Gly
Thr Ser -10 -5 -1 1 5 cag gcc gag gtc cat cct tcc atg acg tgg cag
tcg tgt aca gcg ggt 144Gln Ala Glu Val His Pro Ser Met Thr Trp Gln
Ser Cys Thr Ala Gly 10 15 20 ggt tcg tgt acc aca aac aac ggt aaa
gtc gtg atc gat gca aac tgg 192Gly Ser Cys Thr Thr Asn Asn Gly Lys
Val Val Ile Asp Ala Asn Trp 25 30 35 agg tgg gtg cac aag gtc ggc
gac tac acc aac tgt tac aca ggc aac 240Arg Trp Val His Lys Val Gly
Asp Tyr Thr Asn Cys Tyr Thr Gly Asn 40 45 50 aca tgg gat aca acc
atc tgt ccc gac gat gcc act tgt gca tcc aac 288Thr Trp Asp Thr Thr
Ile Cys Pro Asp Asp Ala Thr Cys Ala Ser Asn 55 60 65 70 tgt gca ctc
gag ggt gcc aac tat gag tcg acg tac gga gtg acc gcc 336Cys Ala Leu
Glu Gly Ala Asn Tyr Glu Ser Thr Tyr Gly Val Thr Ala 75 80 85 tcc
gga aac tcg ctc agg ctc aac ttc gtc aca act tcc cag cag aag 384Ser
Gly Asn Ser Leu Arg Leu Asn Phe Val Thr Thr Ser Gln Gln Lys 90 95
100 aac atc ggc tcg cgg ttg tat atg atg aaa gac gat tcc act tac gag
432Asn Ile Gly Ser Arg Leu Tyr Met Met Lys Asp Asp Ser Thr Tyr Glu
105 110 115 atg ttc aag ctc ctc aac cag gaa ttc act ttc gat gtc gac
gtc tcc 480Met Phe Lys Leu Leu Asn Gln Glu Phe Thr Phe Asp Val Asp
Val Ser 120 125 130 aac ctc cct tgt ggc ttg aac gga gcg ctc tac ttc
gtc gcc atg gat 528Asn Leu Pro Cys Gly Leu Asn Gly Ala Leu Tyr Phe
Val Ala Met Asp 135 140 145 150 gcg gat gga ggc atg tcc aag tat cct
acc aac aaa gca gga gcc aag 576Ala Asp Gly Gly Met Ser Lys Tyr Pro
Thr Asn Lys Ala Gly Ala Lys 155 160 165 tat ggt aca ggt tac tgt gat
tcc cag tgt ccc agg gat ctc aag ttc 624Tyr Gly Thr Gly Tyr Cys Asp
Ser Gln Cys Pro Arg Asp Leu Lys Phe 170 175 180 atc aac ggt cag gcc
aac gtc gag ggt tgg cag cct tcg tcg aac gat 672Ile Asn Gly Gln Ala
Asn Val Glu Gly Trp Gln Pro Ser Ser Asn Asp 185 190 195 gcc aac gca
ggt acc ggc aac cac ggt tcc tgt tgt gcc gaa atg gac 720Ala Asn Ala
Gly Thr Gly Asn His Gly Ser Cys Cys Ala Glu Met Asp 200 205 210 att
tgg gaa gcg aac tcg atc tcg acg gcg ttc act cct cac ccg tgt 768Ile
Trp Glu Ala Asn Ser Ile Ser Thr Ala Phe Thr Pro His Pro Cys 215 220
225 230 gat aca ccc gga cag gtg atg tgt aca ggc gac gcc tgt ggc gga
acc 816Asp Thr Pro Gly Gln Val Met Cys Thr Gly Asp Ala Cys Gly Gly
Thr 235 240 245 tac tcg tcg gat cga tat ggc ggt acg tgt gac ccc gac
ggc tgt gac 864Tyr Ser Ser Asp Arg Tyr Gly Gly Thr Cys Asp Pro Asp
Gly Cys Asp 250 255 260 ttc aac tcc ttc agg cag ggc aac aaa aca ttc
tat gga cct ggc atg 912Phe Asn Ser Phe Arg Gln Gly Asn Lys Thr Phe
Tyr Gly Pro Gly Met 265 270 275 acg gtg gat aca aag tcg aaa ttc aca
gtc gtc act cag ttc atc acc 960Thr Val Asp Thr Lys Ser Lys Phe Thr
Val Val Thr Gln Phe Ile Thr 280 285 290 gac gat ggt acg tcc tcg ggt
acc ttg aag gag atc aaa agg ttc tat 1008Asp Asp Gly Thr Ser Ser Gly
Thr Leu Lys Glu Ile Lys Arg Phe Tyr 295 300 305 310 gtc cag aac gga
aag gtc atc ccg aac tcg gag tcc acg tgg aca gga 1056Val Gln Asn Gly
Lys Val Ile Pro Asn Ser Glu Ser Thr Trp Thr Gly 315 320 325 gtg tcg
ggt aac tcc atc act acg gag tat tgt aca gcc cag aag tcg 1104Val Ser
Gly Asn Ser Ile Thr Thr Glu Tyr Cys Thr Ala Gln Lys Ser 330 335 340
ctc ttc cag gat cag aac gtc ttc gag aaa cat gga ggc ttg gaa gga
1152Leu Phe Gln Asp Gln Asn Val Phe Glu Lys His Gly Gly Leu Glu Gly
345 350 355 atg ggt gcc gca ttg gcc cag ggt atg gtc ctc gtc atg tcc
ttg tgg 1200Met Gly Ala Ala Leu Ala Gln Gly Met Val Leu Val Met Ser
Leu Trp 360 365 370 gac gac cac tcg gcc aac atg ctc tgg ttg gat tcc
aac tac ccc acc 1248Asp Asp His Ser Ala Asn Met Leu Trp Leu Asp Ser
Asn Tyr Pro Thr 375 380 385 390 act gcc gat cct acg aca ccg ggt gtc
gca cgc gga act tgt gat atc 1296Thr Ala Asp Pro Thr Thr Pro Gly Val
Ala Arg Gly Thr Cys Asp Ile 395 400 405 tcc tcg gga gtg cct gca gac
gtc gag gcg aac cat ccc gac gcc tac 1344Ser Ser Gly Val Pro Ala Asp
Val Glu Ala Asn His Pro Asp Ala Tyr 410 415 420 gtg gtc tac tcg aac
att aag gtg gga ccc atc ggt tcg aca ttc aac 1392Val Val Tyr Ser Asn
Ile Lys Val Gly Pro Ile Gly Ser Thr Phe Asn 425 430 435 tcc gga ggc
tcg aac cct gga ggc gga acg acc act act aca acg act 1440Ser Gly Gly
Ser Asn Pro Gly Gly Gly Thr Thr Thr Thr Thr Thr Thr 440 445 450 cag
ccg aca aca aca act acc aca gca ggc aac cct gga ggt aca ggt 1488Gln
Pro Thr Thr Thr Thr Thr Thr Ala Gly Asn Pro Gly Gly Thr Gly 455 460
465 470 gtg gcc cag cac tac gga cag tgt ggc ggt atc gga tgg aca gga
cct 1536Val Ala Gln His Tyr Gly Gln Cys Gly Gly Ile Gly Trp Thr Gly
Pro 475 480 485 act act tgt gca tcg cct tat acc tgt cag aaa ttg aac
gac tac tac 1584Thr Thr Cys Ala Ser Pro Tyr Thr Cys Gln Lys Leu Asn
Asp Tyr Tyr 490 495 500 tcg cag tgt ttg taa 1599Ser Gln Cys Leu 505
71532PRTAspergillus fumigatus 71Met Leu Ala Ser Thr Phe Ser Tyr Arg
Met Tyr Lys Thr Ala Leu Ile -25 -20 -15 Leu Ala Ala Leu Leu Gly Ser
Gly Gln Ala Gln Gln Val Gly Thr Ser -10 -5 -1 1 5 Gln Ala Glu Val
His Pro Ser Met Thr Trp Gln Ser Cys Thr Ala Gly 10 15 20 Gly Ser
Cys Thr Thr Asn Asn Gly Lys Val Val Ile Asp Ala Asn Trp 25 30 35
Arg Trp Val His Lys Val Gly Asp Tyr Thr Asn Cys Tyr Thr Gly Asn 40
45 50 Thr Trp Asp Thr Thr Ile Cys Pro Asp Asp Ala Thr Cys Ala Ser
Asn 55 60 65 70 Cys Ala Leu Glu Gly Ala Asn Tyr Glu Ser Thr Tyr Gly
Val Thr Ala 75 80 85 Ser Gly Asn Ser Leu Arg Leu Asn Phe Val Thr
Thr Ser Gln Gln Lys 90 95 100 Asn Ile Gly Ser Arg Leu Tyr Met Met
Lys Asp Asp Ser Thr Tyr Glu 105 110 115 Met Phe Lys Leu Leu Asn Gln
Glu Phe Thr Phe Asp Val Asp Val Ser 120 125 130 Asn Leu Pro Cys Gly
Leu Asn Gly Ala Leu Tyr Phe Val Ala Met Asp 135 140 145 150 Ala Asp
Gly Gly Met Ser Lys Tyr Pro Thr Asn Lys Ala Gly Ala Lys 155 160 165
Tyr Gly Thr Gly Tyr Cys Asp Ser Gln Cys Pro Arg Asp Leu Lys Phe 170
175 180 Ile Asn Gly Gln Ala Asn Val Glu Gly Trp Gln Pro Ser Ser Asn
Asp 185 190 195 Ala Asn Ala Gly Thr Gly Asn His Gly Ser Cys Cys Ala
Glu Met Asp 200 205 210 Ile Trp Glu Ala Asn Ser Ile Ser Thr Ala Phe
Thr Pro His Pro Cys 215 220 225 230 Asp Thr Pro Gly Gln Val Met Cys
Thr Gly Asp Ala Cys Gly Gly Thr 235 240 245 Tyr Ser Ser Asp Arg Tyr
Gly Gly Thr Cys Asp Pro Asp Gly Cys Asp 250 255 260 Phe Asn Ser Phe
Arg Gln Gly Asn Lys Thr Phe Tyr Gly Pro Gly Met 265 270 275 Thr Val
Asp Thr Lys Ser Lys Phe Thr Val Val Thr Gln Phe Ile Thr 280 285 290
Asp Asp Gly Thr Ser Ser Gly Thr Leu Lys Glu Ile Lys Arg Phe Tyr 295
300 305 310 Val Gln Asn Gly Lys Val Ile Pro Asn Ser Glu Ser Thr Trp
Thr Gly 315 320 325 Val Ser Gly Asn Ser Ile Thr Thr Glu Tyr Cys Thr
Ala Gln Lys Ser 330 335 340 Leu Phe Gln Asp Gln Asn Val Phe Glu Lys
His Gly Gly Leu Glu Gly 345 350 355 Met Gly Ala Ala Leu Ala Gln Gly
Met Val Leu Val Met Ser Leu Trp 360 365 370 Asp Asp His Ser Ala Asn
Met Leu Trp Leu Asp Ser Asn Tyr Pro Thr 375 380 385 390 Thr Ala Asp
Pro Thr Thr Pro Gly Val Ala Arg Gly Thr Cys Asp Ile 395 400 405 Ser
Ser Gly Val Pro Ala Asp Val Glu Ala Asn His Pro Asp Ala Tyr 410 415
420 Val Val Tyr Ser Asn Ile Lys Val Gly Pro Ile Gly Ser Thr Phe Asn
425 430 435 Ser Gly Gly Ser Asn Pro Gly Gly Gly Thr Thr Thr Thr Thr
Thr Thr 440 445 450 Gln Pro Thr Thr Thr Thr Thr Thr Ala Gly Asn Pro
Gly Gly Thr Gly 455 460 465 470 Val Ala Gln His Tyr Gly Gln Cys Gly
Gly Ile Gly Trp Thr Gly Pro 475 480 485 Thr Thr Cys Ala Ser Pro Tyr
Thr Cys Gln Lys Leu Asn Asp Tyr Tyr 490 495 500 Ser Gln Cys Leu 505
721599DNAAspergillus
fumigatussig_peptide(1)..(26)CDS(1)..(1596)mat_peptide(79)..(1596)
72atg ttg gcc tcc acg ttc tcc tat cgc atg tac aaa aca gcg ctc atc
48Met Leu Ala Ser Thr Phe Ser Tyr Arg Met Tyr Lys Thr Ala Leu Ile
-25 -20 -15 ttg gca gcc ctc ttg ggc tcg gga cag gca cag cag gtc gga
acc tcg 96Leu Ala Ala Leu Leu Gly Ser Gly Gln Ala Gln Gln Val Gly
Thr Ser -10 -5 -1 1 5 cag gcc gag gtc cat cct tcc atg acg tgg cag
tcg tgt aca gcg ggt 144Gln Ala Glu Val His Pro Ser Met Thr Trp Gln
Ser Cys Thr Ala Gly 10 15 20 ggt tcg tgt acc aca aac aac ggt aaa
gtc gtg atc gat gca aac tgg 192Gly Ser Cys Thr Thr Asn Asn Gly Lys
Val Val Ile Asp Ala Asn Trp 25 30 35 agg tgg gtg cac aag atc ggc
gac tac acc aac tgt tac aca ggc aac 240Arg Trp Val His Lys Ile Gly
Asp Tyr Thr Asn Cys Tyr Thr Gly Asn 40 45 50 aca tgg gat aca acc
atc tgt ccc gac gat gcc act tgt gca tcc aac 288Thr Trp Asp Thr Thr
Ile Cys Pro Asp Asp Ala Thr Cys Ala Ser Asn 55 60 65 70 tgt gca ctc
gag ggt gcc aac tat gag tcg acg tac gga gtg acc gcc 336Cys Ala Leu
Glu Gly Ala Asn Tyr Glu Ser Thr Tyr Gly Val Thr Ala 75 80 85 tcc
gga aac tcg ctc agg ctc aac ttc gtc aca act tcc cag cag aag 384Ser
Gly Asn Ser Leu Arg Leu Asn Phe Val Thr Thr Ser Gln Gln Lys 90 95
100 aac atc ggc tcg cgg ttg tat atg atg aaa gac gat tcc act tac gag
432Asn Ile Gly Ser Arg Leu Tyr Met Met Lys Asp Asp Ser Thr Tyr Glu
105 110 115 atg ttc aag ctc ctc aac cag gaa ttc act ttc gat gtc gac
gtc tcc 480Met Phe Lys Leu Leu Asn Gln Glu Phe Thr Phe Asp Val Asp
Val Ser 120 125 130 aac ctc cct tgt ggc ttg aac gga gcg ctc tac ttc
gtc gcc atg gat 528Asn Leu Pro Cys Gly Leu Asn Gly Ala Leu Tyr Phe
Val Ala Met Asp 135 140 145 150 gcg gat gga ggc atg tcc aag tat cct
acc aac aaa gca gga gcc aag 576Ala Asp Gly Gly Met Ser Lys Tyr Pro
Thr Asn Lys Ala Gly Ala Lys 155 160 165 tat ggt aca ggt tac tgt gat
tcc cag tgt ccc agg gat ctc aag ttc 624Tyr Gly Thr Gly Tyr Cys Asp
Ser Gln Cys Pro Arg Asp Leu Lys Phe 170 175 180 atc aac ggt cag gcc
aac gtc gag ggt tgg cag cct tcg tcg aac gat 672Ile Asn Gly Gln Ala
Asn Val Glu Gly Trp
Gln Pro Ser Ser Asn Asp 185 190 195 gcc aac gca ggt acc ggc aac cac
ggt tcc tgt tgt gcc gaa atg gac 720Ala Asn Ala Gly Thr Gly Asn His
Gly Ser Cys Cys Ala Glu Met Asp 200 205 210 att tgg gaa gcg aac tcg
atc tcg acg gcg ttc act cct cac ccg tgt 768Ile Trp Glu Ala Asn Ser
Ile Ser Thr Ala Phe Thr Pro His Pro Cys 215 220 225 230 gat aca ccc
gga cag gtg atg tgt aca ggc gac gcc tgt ggc gga acc 816Asp Thr Pro
Gly Gln Val Met Cys Thr Gly Asp Ala Cys Gly Gly Thr 235 240 245 tac
tcg tcg gat cga tat ggc ggt acg tgt gac ccc gac ggc tgt gac 864Tyr
Ser Ser Asp Arg Tyr Gly Gly Thr Cys Asp Pro Asp Gly Cys Asp 250 255
260 ttc aac tcc ttc agg cag ggc aac aaa aca ttc tat gga cct ggc atg
912Phe Asn Ser Phe Arg Gln Gly Asn Lys Thr Phe Tyr Gly Pro Gly Met
265 270 275 acg gtg gat aca aag tcg aaa ttc aca gtc gtc act cag ttc
atc acc 960Thr Val Asp Thr Lys Ser Lys Phe Thr Val Val Thr Gln Phe
Ile Thr 280 285 290 gac gat ggt acg tcc tcg ggt acc ttg aag gag atc
aaa agg ttc tat 1008Asp Asp Gly Thr Ser Ser Gly Thr Leu Lys Glu Ile
Lys Arg Phe Tyr 295 300 305 310 gtc cag aac gga aag gtc atc ccg aac
tcg gag tcc acg tgg aca gga 1056Val Gln Asn Gly Lys Val Ile Pro Asn
Ser Glu Ser Thr Trp Thr Gly 315 320 325 gtg tcg ggt aac tcc atc act
acg gag tat tgt aca gcc cag aag tcg 1104Val Ser Gly Asn Ser Ile Thr
Thr Glu Tyr Cys Thr Ala Gln Lys Ser 330 335 340 ctc ttc cag gat cag
aac gtc ttc gag aaa cat gga ggc ttg gaa gga 1152Leu Phe Gln Asp Gln
Asn Val Phe Glu Lys His Gly Gly Leu Glu Gly 345 350 355 atg ggt gcc
gca ttg gcc cag ggt atg gtc ctc gtc atg tcc ttg tgg 1200Met Gly Ala
Ala Leu Ala Gln Gly Met Val Leu Val Met Ser Leu Trp 360 365 370 gac
gac cac tcg gcc aac atg ctc tgg ttg gat tcc aac tac ccc acc 1248Asp
Asp His Ser Ala Asn Met Leu Trp Leu Asp Ser Asn Tyr Pro Thr 375 380
385 390 act gcc tcg tcc acg aca ccg ggt gtc gca cgc gga act tgt gat
atc 1296Thr Ala Ser Ser Thr Thr Pro Gly Val Ala Arg Gly Thr Cys Asp
Ile 395 400 405 tcc tcg gga gtg cct gca gac gtc gag gcg aac cat ccc
gac gcc tac 1344Ser Ser Gly Val Pro Ala Asp Val Glu Ala Asn His Pro
Asp Ala Tyr 410 415 420 gtg gtc tac tcg aac att aag gtg gga ccc atc
ggt tcg aca ttc aac 1392Val Val Tyr Ser Asn Ile Lys Val Gly Pro Ile
Gly Ser Thr Phe Asn 425 430 435 tcc gga ggc tcg aac cct gga ggc gga
acg acc act act aca acg act 1440Ser Gly Gly Ser Asn Pro Gly Gly Gly
Thr Thr Thr Thr Thr Thr Thr 440 445 450 cag ccg aca aca aca act acc
aca gca ggc aac cct gga ggt aca ggt 1488Gln Pro Thr Thr Thr Thr Thr
Thr Ala Gly Asn Pro Gly Gly Thr Gly 455 460 465 470 gtg gcc cag cac
tac gga cag tgt ggc ggt atc gga tgg aca gga cct 1536Val Ala Gln His
Tyr Gly Gln Cys Gly Gly Ile Gly Trp Thr Gly Pro 475 480 485 act act
tgt gca tcg cct tat acc tgt cag aaa ttg aac gac tac tac 1584Thr Thr
Cys Ala Ser Pro Tyr Thr Cys Gln Lys Leu Asn Asp Tyr Tyr 490 495 500
tcg cag tgt ttg taa 1599Ser Gln Cys Leu 505 73532PRTAspergillus
fumigatus 73Met Leu Ala Ser Thr Phe Ser Tyr Arg Met Tyr Lys Thr Ala
Leu Ile -25 -20 -15 Leu Ala Ala Leu Leu Gly Ser Gly Gln Ala Gln Gln
Val Gly Thr Ser -10 -5 -1 1 5 Gln Ala Glu Val His Pro Ser Met Thr
Trp Gln Ser Cys Thr Ala Gly 10 15 20 Gly Ser Cys Thr Thr Asn Asn
Gly Lys Val Val Ile Asp Ala Asn Trp 25 30 35 Arg Trp Val His Lys
Ile Gly Asp Tyr Thr Asn Cys Tyr Thr Gly Asn 40 45 50 Thr Trp Asp
Thr Thr Ile Cys Pro Asp Asp Ala Thr Cys Ala Ser Asn 55 60 65 70 Cys
Ala Leu Glu Gly Ala Asn Tyr Glu Ser Thr Tyr Gly Val Thr Ala 75 80
85 Ser Gly Asn Ser Leu Arg Leu Asn Phe Val Thr Thr Ser Gln Gln Lys
90 95 100 Asn Ile Gly Ser Arg Leu Tyr Met Met Lys Asp Asp Ser Thr
Tyr Glu 105 110 115 Met Phe Lys Leu Leu Asn Gln Glu Phe Thr Phe Asp
Val Asp Val Ser 120 125 130 Asn Leu Pro Cys Gly Leu Asn Gly Ala Leu
Tyr Phe Val Ala Met Asp 135 140 145 150 Ala Asp Gly Gly Met Ser Lys
Tyr Pro Thr Asn Lys Ala Gly Ala Lys 155 160 165 Tyr Gly Thr Gly Tyr
Cys Asp Ser Gln Cys Pro Arg Asp Leu Lys Phe 170 175 180 Ile Asn Gly
Gln Ala Asn Val Glu Gly Trp Gln Pro Ser Ser Asn Asp 185 190 195 Ala
Asn Ala Gly Thr Gly Asn His Gly Ser Cys Cys Ala Glu Met Asp 200 205
210 Ile Trp Glu Ala Asn Ser Ile Ser Thr Ala Phe Thr Pro His Pro Cys
215 220 225 230 Asp Thr Pro Gly Gln Val Met Cys Thr Gly Asp Ala Cys
Gly Gly Thr 235 240 245 Tyr Ser Ser Asp Arg Tyr Gly Gly Thr Cys Asp
Pro Asp Gly Cys Asp 250 255 260 Phe Asn Ser Phe Arg Gln Gly Asn Lys
Thr Phe Tyr Gly Pro Gly Met 265 270 275 Thr Val Asp Thr Lys Ser Lys
Phe Thr Val Val Thr Gln Phe Ile Thr 280 285 290 Asp Asp Gly Thr Ser
Ser Gly Thr Leu Lys Glu Ile Lys Arg Phe Tyr 295 300 305 310 Val Gln
Asn Gly Lys Val Ile Pro Asn Ser Glu Ser Thr Trp Thr Gly 315 320 325
Val Ser Gly Asn Ser Ile Thr Thr Glu Tyr Cys Thr Ala Gln Lys Ser 330
335 340 Leu Phe Gln Asp Gln Asn Val Phe Glu Lys His Gly Gly Leu Glu
Gly 345 350 355 Met Gly Ala Ala Leu Ala Gln Gly Met Val Leu Val Met
Ser Leu Trp 360 365 370 Asp Asp His Ser Ala Asn Met Leu Trp Leu Asp
Ser Asn Tyr Pro Thr 375 380 385 390 Thr Ala Ser Ser Thr Thr Pro Gly
Val Ala Arg Gly Thr Cys Asp Ile 395 400 405 Ser Ser Gly Val Pro Ala
Asp Val Glu Ala Asn His Pro Asp Ala Tyr 410 415 420 Val Val Tyr Ser
Asn Ile Lys Val Gly Pro Ile Gly Ser Thr Phe Asn 425 430 435 Ser Gly
Gly Ser Asn Pro Gly Gly Gly Thr Thr Thr Thr Thr Thr Thr 440 445 450
Gln Pro Thr Thr Thr Thr Thr Thr Ala Gly Asn Pro Gly Gly Thr Gly 455
460 465 470 Val Ala Gln His Tyr Gly Gln Cys Gly Gly Ile Gly Trp Thr
Gly Pro 475 480 485 Thr Thr Cys Ala Ser Pro Tyr Thr Cys Gln Lys Leu
Asn Asp Tyr Tyr 490 495 500 Ser Gln Cys Leu 505
741599DNAAspergillus
fumigatussig_peptide(1)..(26)CDS(1)..(1596)mat_peptide(79)..(1596)
74atg ttg gcc tcc acg ttc tcc tat cgc atg tac aaa aca gcg ctc atc
48Met Leu Ala Ser Thr Phe Ser Tyr Arg Met Tyr Lys Thr Ala Leu Ile
-25 -20 -15 ttg gca gcc ctc ttg ggc tcg gga cag gca cag cag gtc gga
acc tcg 96Leu Ala Ala Leu Leu Gly Ser Gly Gln Ala Gln Gln Val Gly
Thr Ser -10 -5 -1 1 5 cag gcc gag gtc cat cct tcc atg acg tgg cag
tcg tgt aca gcg ggt 144Gln Ala Glu Val His Pro Ser Met Thr Trp Gln
Ser Cys Thr Ala Gly 10 15 20 ggt tcg tgt acc aca aac aac ggt aaa
gtc gtg atc gat gca aac tgg 192Gly Ser Cys Thr Thr Asn Asn Gly Lys
Val Val Ile Asp Ala Asn Trp 25 30 35 agg tgg gtg cac aag atg ggc
gac tac acc aac tgt tac aca ggc aac 240Arg Trp Val His Lys Met Gly
Asp Tyr Thr Asn Cys Tyr Thr Gly Asn 40 45 50 aca tgg gat aca acc
atc tgt ccc gac gat gcc act tgt gca tcc aac 288Thr Trp Asp Thr Thr
Ile Cys Pro Asp Asp Ala Thr Cys Ala Ser Asn 55 60 65 70 tgt gca ctc
gag ggt gcc aac tat gag tcg acg tac gga gtg acc gcc 336Cys Ala Leu
Glu Gly Ala Asn Tyr Glu Ser Thr Tyr Gly Val Thr Ala 75 80 85 tcc
gga aac tcg ctc agg ctc aac ttc gtc aca act tcc cag cag aag 384Ser
Gly Asn Ser Leu Arg Leu Asn Phe Val Thr Thr Ser Gln Gln Lys 90 95
100 aac atc ggc tcg cgg ttg tat atg atg aaa gac gat tcc act tac gag
432Asn Ile Gly Ser Arg Leu Tyr Met Met Lys Asp Asp Ser Thr Tyr Glu
105 110 115 atg ttc aag ctc ctc aac cag gaa ttc act ttc gat gtc gac
gtc tcc 480Met Phe Lys Leu Leu Asn Gln Glu Phe Thr Phe Asp Val Asp
Val Ser 120 125 130 aac ctc cct tgt ggc ttg aac gga gcg ctc tac ttc
gtc gcc atg gat 528Asn Leu Pro Cys Gly Leu Asn Gly Ala Leu Tyr Phe
Val Ala Met Asp 135 140 145 150 gcg gat gga ggc atg tcc aag tat cct
acc aac aaa gca gga gcc aag 576Ala Asp Gly Gly Met Ser Lys Tyr Pro
Thr Asn Lys Ala Gly Ala Lys 155 160 165 tat ggt aca ggt tac tgt gat
tcc cag tgt ccc agg gat ctc aag ttc 624Tyr Gly Thr Gly Tyr Cys Asp
Ser Gln Cys Pro Arg Asp Leu Lys Phe 170 175 180 atc aac ggt cag gcc
aac gtc gag ggt tgg cag cct tcg tcg aac gat 672Ile Asn Gly Gln Ala
Asn Val Glu Gly Trp Gln Pro Ser Ser Asn Asp 185 190 195 gcc aac gca
ggt acc ggc aac cac ggt tcc tgt tgt gcc gaa atg gac 720Ala Asn Ala
Gly Thr Gly Asn His Gly Ser Cys Cys Ala Glu Met Asp 200 205 210 att
tgg gaa gcg aac tcg atc tcg acg gcg ttc act cct cac ccg tgt 768Ile
Trp Glu Ala Asn Ser Ile Ser Thr Ala Phe Thr Pro His Pro Cys 215 220
225 230 gat aca ccc gga cag gtg atg tgt aca ggc gac gcc tgt ggc gga
acc 816Asp Thr Pro Gly Gln Val Met Cys Thr Gly Asp Ala Cys Gly Gly
Thr 235 240 245 tac tcg tcg gat cga tat ggc ggt acg tgt gac ccc gac
ggc tgt gac 864Tyr Ser Ser Asp Arg Tyr Gly Gly Thr Cys Asp Pro Asp
Gly Cys Asp 250 255 260 ttc aac tcc ttc agg cag ggc aac aaa aca ttc
tat gga cct ggc atg 912Phe Asn Ser Phe Arg Gln Gly Asn Lys Thr Phe
Tyr Gly Pro Gly Met 265 270 275 acg gtg gat aca aag tcg aaa ttc aca
gtc gtc act cag ttc atc acc 960Thr Val Asp Thr Lys Ser Lys Phe Thr
Val Val Thr Gln Phe Ile Thr 280 285 290 gac gat ggt acg tcc tcg ggt
acc ttg aag gag atc aaa agg ttc tat 1008Asp Asp Gly Thr Ser Ser Gly
Thr Leu Lys Glu Ile Lys Arg Phe Tyr 295 300 305 310 gtc cag aac gga
aag gtc atc ccg aac tcg gag tcc acg tgg aca gga 1056Val Gln Asn Gly
Lys Val Ile Pro Asn Ser Glu Ser Thr Trp Thr Gly 315 320 325 gtg tcg
ggt aac tcc atc act acg gag tat tgt aca gcc cag aag tcg 1104Val Ser
Gly Asn Ser Ile Thr Thr Glu Tyr Cys Thr Ala Gln Lys Ser 330 335 340
ctc ttc cag gat cag aac gtc ttc gag aaa cat gga ggc ttg gaa gga
1152Leu Phe Gln Asp Gln Asn Val Phe Glu Lys His Gly Gly Leu Glu Gly
345 350 355 atg ggt gcc gca ttg gcc cag ggt atg gtc ctc gtc atg tcc
ttg tgg 1200Met Gly Ala Ala Leu Ala Gln Gly Met Val Leu Val Met Ser
Leu Trp 360 365 370 gac gac cac tcg gcc aac atg ctc tgg ttg gat tcc
aac tac ccc acc 1248Asp Asp His Ser Ala Asn Met Leu Trp Leu Asp Ser
Asn Tyr Pro Thr 375 380 385 390 act gcc tcg tcc acg aca ccg ggt gtc
gca cgc gga act tgt gat atc 1296Thr Ala Ser Ser Thr Thr Pro Gly Val
Ala Arg Gly Thr Cys Asp Ile 395 400 405 tcc tcg gga gtg cct gca gac
gtc gag gcg aac cat ccc gac gcc tac 1344Ser Ser Gly Val Pro Ala Asp
Val Glu Ala Asn His Pro Asp Ala Tyr 410 415 420 gtg gtc tac tcg aac
att aag gtg gga ccc atc ggt tcg aca ttc aac 1392Val Val Tyr Ser Asn
Ile Lys Val Gly Pro Ile Gly Ser Thr Phe Asn 425 430 435 tcc gga ggc
tcg aac cct gga ggc gga acg acc act act aca acg act 1440Ser Gly Gly
Ser Asn Pro Gly Gly Gly Thr Thr Thr Thr Thr Thr Thr 440 445 450 cag
ccg aca aca aca act acc aca gca ggc aac cct gga ggt aca ggt 1488Gln
Pro Thr Thr Thr Thr Thr Thr Ala Gly Asn Pro Gly Gly Thr Gly 455 460
465 470 gtg gcc cag cac tac gga cag tgt ggc ggt atc gga tgg aca gga
cct 1536Val Ala Gln His Tyr Gly Gln Cys Gly Gly Ile Gly Trp Thr Gly
Pro 475 480 485 act act tgt gca tcg cct tat acc tgt cag aaa ttg aac
gac tac tac 1584Thr Thr Cys Ala Ser Pro Tyr Thr Cys Gln Lys Leu Asn
Asp Tyr Tyr 490 495 500 tcg cag tgt ttg taa 1599Ser Gln Cys Leu 505
75532PRTAspergillus fumigatus 75Met Leu Ala Ser Thr Phe Ser Tyr Arg
Met Tyr Lys Thr Ala Leu Ile -25 -20 -15 Leu Ala Ala Leu Leu Gly Ser
Gly Gln Ala Gln Gln Val Gly Thr Ser -10 -5 -1 1 5 Gln Ala Glu Val
His Pro Ser Met Thr Trp Gln Ser Cys Thr Ala Gly 10 15 20 Gly Ser
Cys Thr Thr Asn Asn Gly Lys Val Val Ile Asp Ala Asn Trp 25 30 35
Arg Trp Val His Lys Met Gly Asp Tyr Thr Asn Cys Tyr Thr Gly Asn 40
45 50 Thr Trp Asp Thr Thr Ile Cys Pro Asp Asp Ala Thr Cys Ala Ser
Asn 55 60 65 70 Cys Ala Leu Glu Gly Ala Asn Tyr Glu Ser Thr Tyr Gly
Val Thr Ala 75 80 85 Ser Gly Asn Ser Leu Arg Leu Asn Phe Val Thr
Thr Ser Gln Gln Lys 90 95 100 Asn Ile Gly Ser Arg Leu Tyr Met Met
Lys Asp Asp Ser Thr Tyr Glu 105 110 115 Met Phe Lys Leu Leu Asn Gln
Glu Phe Thr Phe Asp Val Asp Val Ser 120 125 130 Asn Leu Pro Cys Gly
Leu Asn Gly Ala Leu Tyr Phe Val Ala Met Asp 135 140 145 150 Ala Asp
Gly Gly Met Ser Lys Tyr Pro Thr Asn Lys Ala Gly Ala Lys 155 160 165
Tyr Gly Thr Gly Tyr Cys Asp Ser Gln Cys Pro Arg Asp Leu Lys Phe 170
175 180 Ile Asn Gly Gln Ala Asn Val Glu Gly Trp Gln Pro Ser Ser Asn
Asp 185 190 195 Ala Asn Ala Gly Thr Gly Asn His Gly Ser Cys Cys Ala
Glu Met Asp 200 205 210 Ile Trp Glu Ala Asn Ser Ile Ser Thr Ala Phe
Thr Pro His Pro Cys 215 220 225 230 Asp Thr Pro Gly Gln Val Met Cys
Thr Gly Asp
Ala Cys Gly Gly Thr 235 240 245 Tyr Ser Ser Asp Arg Tyr Gly Gly Thr
Cys Asp Pro Asp Gly Cys Asp 250 255 260 Phe Asn Ser Phe Arg Gln Gly
Asn Lys Thr Phe Tyr Gly Pro Gly Met 265 270 275 Thr Val Asp Thr Lys
Ser Lys Phe Thr Val Val Thr Gln Phe Ile Thr 280 285 290 Asp Asp Gly
Thr Ser Ser Gly Thr Leu Lys Glu Ile Lys Arg Phe Tyr 295 300 305 310
Val Gln Asn Gly Lys Val Ile Pro Asn Ser Glu Ser Thr Trp Thr Gly 315
320 325 Val Ser Gly Asn Ser Ile Thr Thr Glu Tyr Cys Thr Ala Gln Lys
Ser 330 335 340 Leu Phe Gln Asp Gln Asn Val Phe Glu Lys His Gly Gly
Leu Glu Gly 345 350 355 Met Gly Ala Ala Leu Ala Gln Gly Met Val Leu
Val Met Ser Leu Trp 360 365 370 Asp Asp His Ser Ala Asn Met Leu Trp
Leu Asp Ser Asn Tyr Pro Thr 375 380 385 390 Thr Ala Ser Ser Thr Thr
Pro Gly Val Ala Arg Gly Thr Cys Asp Ile 395 400 405 Ser Ser Gly Val
Pro Ala Asp Val Glu Ala Asn His Pro Asp Ala Tyr 410 415 420 Val Val
Tyr Ser Asn Ile Lys Val Gly Pro Ile Gly Ser Thr Phe Asn 425 430 435
Ser Gly Gly Ser Asn Pro Gly Gly Gly Thr Thr Thr Thr Thr Thr Thr 440
445 450 Gln Pro Thr Thr Thr Thr Thr Thr Ala Gly Asn Pro Gly Gly Thr
Gly 455 460 465 470 Val Ala Gln His Tyr Gly Gln Cys Gly Gly Ile Gly
Trp Thr Gly Pro 475 480 485 Thr Thr Cys Ala Ser Pro Tyr Thr Cys Gln
Lys Leu Asn Asp Tyr Tyr 490 495 500 Ser Gln Cys Leu 505
761599DNAAspergillus
fumigatussig_peptide(1)..(26)CDS(1)..(1596)mat_peptide(79)..(1596)
76atg ttg gcc tcc acg ttc tcc tat cgc atg tac aaa aca gcg ctc atc
48Met Leu Ala Ser Thr Phe Ser Tyr Arg Met Tyr Lys Thr Ala Leu Ile
-25 -20 -15 ttg gca gcc ctc ttg ggc tcg gga cag gca cag cag gtc gga
acc tcg 96Leu Ala Ala Leu Leu Gly Ser Gly Gln Ala Gln Gln Val Gly
Thr Ser -10 -5 -1 1 5 cag gcc gag gtc cat cct tcc atg acg tgg cag
tcg tgt aca gcg ggt 144Gln Ala Glu Val His Pro Ser Met Thr Trp Gln
Ser Cys Thr Ala Gly 10 15 20 ggt tcg tgt acc aca aac aac ggt aaa
gtc gtg atc gat gca aac tgg 192Gly Ser Cys Thr Thr Asn Asn Gly Lys
Val Val Ile Asp Ala Asn Trp 25 30 35 agg tgg gtg cac aag aac ggc
gac tac acc aac tgt tac aca ggc aac 240Arg Trp Val His Lys Asn Gly
Asp Tyr Thr Asn Cys Tyr Thr Gly Asn 40 45 50 aca tgg gat aca acc
atc tgt ccc gac gat gcc act tgt gca tcc aac 288Thr Trp Asp Thr Thr
Ile Cys Pro Asp Asp Ala Thr Cys Ala Ser Asn 55 60 65 70 tgt gca ctc
gag ggt gcc aac tat gag tcg acg tac gga gtg acc gcc 336Cys Ala Leu
Glu Gly Ala Asn Tyr Glu Ser Thr Tyr Gly Val Thr Ala 75 80 85 tcc
gga aac tcg ctc agg ctc aac ttc gtc aca act tcc cag cag aag 384Ser
Gly Asn Ser Leu Arg Leu Asn Phe Val Thr Thr Ser Gln Gln Lys 90 95
100 aac atc ggc tcg cgg ttg tat atg atg aaa gac gat tcc act tac gag
432Asn Ile Gly Ser Arg Leu Tyr Met Met Lys Asp Asp Ser Thr Tyr Glu
105 110 115 atg ttc aag ctc ctc aac cag gaa ttc act ttc gat gtc gac
gtc tcc 480Met Phe Lys Leu Leu Asn Gln Glu Phe Thr Phe Asp Val Asp
Val Ser 120 125 130 aac ctc cct tgt ggc ttg aac gga gcg ctc tac ttc
gtc gcc atg gat 528Asn Leu Pro Cys Gly Leu Asn Gly Ala Leu Tyr Phe
Val Ala Met Asp 135 140 145 150 gcg gat gga ggc atg tcc aag tat cct
acc aac aaa gca gga gcc aag 576Ala Asp Gly Gly Met Ser Lys Tyr Pro
Thr Asn Lys Ala Gly Ala Lys 155 160 165 tat ggt aca ggt tac tgt gat
tcc cag tgt ccc agg gat ctc aag ttc 624Tyr Gly Thr Gly Tyr Cys Asp
Ser Gln Cys Pro Arg Asp Leu Lys Phe 170 175 180 atc aac ggt cag gcc
aac gtc gag ggt tgg cag cct tcg tcg aac gat 672Ile Asn Gly Gln Ala
Asn Val Glu Gly Trp Gln Pro Ser Ser Asn Asp 185 190 195 gcc aac gca
ggt acc ggc aac cac ggt tcc tgt tgt gcc gaa atg gac 720Ala Asn Ala
Gly Thr Gly Asn His Gly Ser Cys Cys Ala Glu Met Asp 200 205 210 att
tgg gaa gcg aac tcg atc tcg acg gcg ttc act cct cac ccg tgt 768Ile
Trp Glu Ala Asn Ser Ile Ser Thr Ala Phe Thr Pro His Pro Cys 215 220
225 230 gat aca ccc gga cag gtg atg tgt aca ggc gac gcc tgt ggc gga
acc 816Asp Thr Pro Gly Gln Val Met Cys Thr Gly Asp Ala Cys Gly Gly
Thr 235 240 245 tac tcg tcg gat cga tat ggc ggt acg tgt gac ccc gac
ggc tgt gac 864Tyr Ser Ser Asp Arg Tyr Gly Gly Thr Cys Asp Pro Asp
Gly Cys Asp 250 255 260 ttc aac tcc ttc agg cag ggc aac aaa aca ttc
tat gga cct ggc atg 912Phe Asn Ser Phe Arg Gln Gly Asn Lys Thr Phe
Tyr Gly Pro Gly Met 265 270 275 acg gtg gat aca aag tcg aaa ttc aca
gtc gtc act cag ttc atc acc 960Thr Val Asp Thr Lys Ser Lys Phe Thr
Val Val Thr Gln Phe Ile Thr 280 285 290 gac gat ggt acg tcc tcg ggt
acc ttg aag gag atc aaa agg ttc tat 1008Asp Asp Gly Thr Ser Ser Gly
Thr Leu Lys Glu Ile Lys Arg Phe Tyr 295 300 305 310 gtc cag aac gga
aag gtc atc ccg aac tcg gag tcc acg tgg aca gga 1056Val Gln Asn Gly
Lys Val Ile Pro Asn Ser Glu Ser Thr Trp Thr Gly 315 320 325 gtg tcg
ggt aac tcc atc act acg gag tat tgt aca gcc cag aag tcg 1104Val Ser
Gly Asn Ser Ile Thr Thr Glu Tyr Cys Thr Ala Gln Lys Ser 330 335 340
ctc ttc cag gat cag aac gtc ttc gag aaa cat gga ggc ttg gaa gga
1152Leu Phe Gln Asp Gln Asn Val Phe Glu Lys His Gly Gly Leu Glu Gly
345 350 355 atg ggt gcc gca ttg gcc cag ggt atg gtc ctc gtc atg tcc
ttg tgg 1200Met Gly Ala Ala Leu Ala Gln Gly Met Val Leu Val Met Ser
Leu Trp 360 365 370 gac gac cac tcg gcc aac atg ctc tgg ttg gat tcc
aac tac ccc acc 1248Asp Asp His Ser Ala Asn Met Leu Trp Leu Asp Ser
Asn Tyr Pro Thr 375 380 385 390 act gcc tcg tcc acg aca ccg ggt gtc
gca cgc gga act tgt gat atc 1296Thr Ala Ser Ser Thr Thr Pro Gly Val
Ala Arg Gly Thr Cys Asp Ile 395 400 405 tcc tcg gga gtg cct gca gac
gtc gag gcg aac cat ccc gac gcc tac 1344Ser Ser Gly Val Pro Ala Asp
Val Glu Ala Asn His Pro Asp Ala Tyr 410 415 420 gtg gtc tac tcg aac
att aag gtg gga ccc atc ggt tcg aca ttc aac 1392Val Val Tyr Ser Asn
Ile Lys Val Gly Pro Ile Gly Ser Thr Phe Asn 425 430 435 tcc gga ggc
tcg aac cct gga ggc gga acg acc act act aca acg act 1440Ser Gly Gly
Ser Asn Pro Gly Gly Gly Thr Thr Thr Thr Thr Thr Thr 440 445 450 cag
ccg aca aca aca act acc aca gca ggc aac cct gga ggt aca ggt 1488Gln
Pro Thr Thr Thr Thr Thr Thr Ala Gly Asn Pro Gly Gly Thr Gly 455 460
465 470 gtg gcc cag cac tac gga cag tgt ggc ggt atc gga tgg aca gga
cct 1536Val Ala Gln His Tyr Gly Gln Cys Gly Gly Ile Gly Trp Thr Gly
Pro 475 480 485 act act tgt gca tcg cct tat acc tgt cag aaa ttg aac
gac tac tac 1584Thr Thr Cys Ala Ser Pro Tyr Thr Cys Gln Lys Leu Asn
Asp Tyr Tyr 490 495 500 tcg cag tgt ttg taa 1599Ser Gln Cys Leu 505
77532PRTAspergillus fumigatus 77Met Leu Ala Ser Thr Phe Ser Tyr Arg
Met Tyr Lys Thr Ala Leu Ile -25 -20 -15 Leu Ala Ala Leu Leu Gly Ser
Gly Gln Ala Gln Gln Val Gly Thr Ser -10 -5 -1 1 5 Gln Ala Glu Val
His Pro Ser Met Thr Trp Gln Ser Cys Thr Ala Gly 10 15 20 Gly Ser
Cys Thr Thr Asn Asn Gly Lys Val Val Ile Asp Ala Asn Trp 25 30 35
Arg Trp Val His Lys Asn Gly Asp Tyr Thr Asn Cys Tyr Thr Gly Asn 40
45 50 Thr Trp Asp Thr Thr Ile Cys Pro Asp Asp Ala Thr Cys Ala Ser
Asn 55 60 65 70 Cys Ala Leu Glu Gly Ala Asn Tyr Glu Ser Thr Tyr Gly
Val Thr Ala 75 80 85 Ser Gly Asn Ser Leu Arg Leu Asn Phe Val Thr
Thr Ser Gln Gln Lys 90 95 100 Asn Ile Gly Ser Arg Leu Tyr Met Met
Lys Asp Asp Ser Thr Tyr Glu 105 110 115 Met Phe Lys Leu Leu Asn Gln
Glu Phe Thr Phe Asp Val Asp Val Ser 120 125 130 Asn Leu Pro Cys Gly
Leu Asn Gly Ala Leu Tyr Phe Val Ala Met Asp 135 140 145 150 Ala Asp
Gly Gly Met Ser Lys Tyr Pro Thr Asn Lys Ala Gly Ala Lys 155 160 165
Tyr Gly Thr Gly Tyr Cys Asp Ser Gln Cys Pro Arg Asp Leu Lys Phe 170
175 180 Ile Asn Gly Gln Ala Asn Val Glu Gly Trp Gln Pro Ser Ser Asn
Asp 185 190 195 Ala Asn Ala Gly Thr Gly Asn His Gly Ser Cys Cys Ala
Glu Met Asp 200 205 210 Ile Trp Glu Ala Asn Ser Ile Ser Thr Ala Phe
Thr Pro His Pro Cys 215 220 225 230 Asp Thr Pro Gly Gln Val Met Cys
Thr Gly Asp Ala Cys Gly Gly Thr 235 240 245 Tyr Ser Ser Asp Arg Tyr
Gly Gly Thr Cys Asp Pro Asp Gly Cys Asp 250 255 260 Phe Asn Ser Phe
Arg Gln Gly Asn Lys Thr Phe Tyr Gly Pro Gly Met 265 270 275 Thr Val
Asp Thr Lys Ser Lys Phe Thr Val Val Thr Gln Phe Ile Thr 280 285 290
Asp Asp Gly Thr Ser Ser Gly Thr Leu Lys Glu Ile Lys Arg Phe Tyr 295
300 305 310 Val Gln Asn Gly Lys Val Ile Pro Asn Ser Glu Ser Thr Trp
Thr Gly 315 320 325 Val Ser Gly Asn Ser Ile Thr Thr Glu Tyr Cys Thr
Ala Gln Lys Ser 330 335 340 Leu Phe Gln Asp Gln Asn Val Phe Glu Lys
His Gly Gly Leu Glu Gly 345 350 355 Met Gly Ala Ala Leu Ala Gln Gly
Met Val Leu Val Met Ser Leu Trp 360 365 370 Asp Asp His Ser Ala Asn
Met Leu Trp Leu Asp Ser Asn Tyr Pro Thr 375 380 385 390 Thr Ala Ser
Ser Thr Thr Pro Gly Val Ala Arg Gly Thr Cys Asp Ile 395 400 405 Ser
Ser Gly Val Pro Ala Asp Val Glu Ala Asn His Pro Asp Ala Tyr 410 415
420 Val Val Tyr Ser Asn Ile Lys Val Gly Pro Ile Gly Ser Thr Phe Asn
425 430 435 Ser Gly Gly Ser Asn Pro Gly Gly Gly Thr Thr Thr Thr Thr
Thr Thr 440 445 450 Gln Pro Thr Thr Thr Thr Thr Thr Ala Gly Asn Pro
Gly Gly Thr Gly 455 460 465 470 Val Ala Gln His Tyr Gly Gln Cys Gly
Gly Ile Gly Trp Thr Gly Pro 475 480 485 Thr Thr Cys Ala Ser Pro Tyr
Thr Cys Gln Lys Leu Asn Asp Tyr Tyr 490 495 500 Ser Gln Cys Leu 505
781599DNAAspergillus
fumigatussig_peptide(1)..(26)CDS(1)..(1596)mat_peptide(79)..(1596)
78atg ttg gcc tcc acg ttc tcc tat cgc atg tac aaa aca gcg ctc atc
48Met Leu Ala Ser Thr Phe Ser Tyr Arg Met Tyr Lys Thr Ala Leu Ile
-25 -20 -15 ttg gca gcc ctc ttg ggc tcg gga cag gca cag cag gtc gga
acc tcg 96Leu Ala Ala Leu Leu Gly Ser Gly Gln Ala Gln Gln Val Gly
Thr Ser -10 -5 -1 1 5 cag gcc gag gtc cat cct tcc atg acg tgg cag
tcg tgt aca gcg ggt 144Gln Ala Glu Val His Pro Ser Met Thr Trp Gln
Ser Cys Thr Ala Gly 10 15 20 ggt tcg tgt acc aca aac aac ggt aaa
gtc gtg atc gat gca aac tgg 192Gly Ser Cys Thr Thr Asn Asn Gly Lys
Val Val Ile Asp Ala Asn Trp 25 30 35 agg tgg gtg cac aag aag ggc
gac tac acc aac tgt tac aca ggc aac 240Arg Trp Val His Lys Lys Gly
Asp Tyr Thr Asn Cys Tyr Thr Gly Asn 40 45 50 aca tgg gat aca acc
atc tgt ccc gac gat gcc act tgt gca tcc aac 288Thr Trp Asp Thr Thr
Ile Cys Pro Asp Asp Ala Thr Cys Ala Ser Asn 55 60 65 70 tgt gca ctc
gag ggt gcc aac tat gag tcg acg tac gga gtg acc gcc 336Cys Ala Leu
Glu Gly Ala Asn Tyr Glu Ser Thr Tyr Gly Val Thr Ala 75 80 85 tcc
gga aac tcg ctc agg ctc aac ttc gtc aca act tcc cag cag aag 384Ser
Gly Asn Ser Leu Arg Leu Asn Phe Val Thr Thr Ser Gln Gln Lys 90 95
100 aac atc ggc tcg cgg ttg tat atg atg aaa gac gat tcc act tac gag
432Asn Ile Gly Ser Arg Leu Tyr Met Met Lys Asp Asp Ser Thr Tyr Glu
105 110 115 atg ttc aag ctc ctc aac cag gaa ttc act ttc gat gtc gac
gtc tcc 480Met Phe Lys Leu Leu Asn Gln Glu Phe Thr Phe Asp Val Asp
Val Ser 120 125 130 aac ctc cct tgt ggc ttg aac gga gcg ctc tac ttc
gtc gcc atg gat 528Asn Leu Pro Cys Gly Leu Asn Gly Ala Leu Tyr Phe
Val Ala Met Asp 135 140 145 150 gcg gat gga ggc atg tcc aag tat cct
acc aac aaa gca gga gcc aag 576Ala Asp Gly Gly Met Ser Lys Tyr Pro
Thr Asn Lys Ala Gly Ala Lys 155 160 165 tat ggt aca ggt tac tgt gat
tcc cag tgt ccc agg gat ctc aag ttc 624Tyr Gly Thr Gly Tyr Cys Asp
Ser Gln Cys Pro Arg Asp Leu Lys Phe 170 175 180 atc aac ggt cag gcc
aac gtc gag ggt tgg cag cct tcg tcg aac gat 672Ile Asn Gly Gln Ala
Asn Val Glu Gly Trp Gln Pro Ser Ser Asn Asp 185 190 195 gcc aac gca
ggt acc ggc aac cac ggt tcc tgt tgt gcc gaa atg gac 720Ala Asn Ala
Gly Thr Gly Asn His Gly Ser Cys Cys Ala Glu Met Asp 200 205 210 att
tgg gaa gcg aac tcg atc tcg acg gcg ttc act cct cac ccg tgt 768Ile
Trp Glu Ala Asn Ser Ile Ser Thr Ala Phe Thr Pro His Pro Cys 215 220
225 230 gat aca ccc gga cag gtg atg tgt aca ggc gac gcc tgt ggc gga
acc 816Asp Thr Pro Gly Gln Val Met Cys Thr Gly Asp Ala Cys Gly Gly
Thr 235 240 245 tac tcg tcg gat cga tat ggc ggt acg tgt gac ccc gac
ggc tgt gac 864Tyr Ser Ser Asp Arg Tyr Gly Gly Thr Cys Asp Pro Asp
Gly Cys Asp 250 255 260 ttc aac tcc ttc agg cag ggc aac aaa aca ttc
tat gga cct ggc atg 912Phe Asn Ser Phe Arg Gln Gly Asn Lys Thr Phe
Tyr Gly Pro Gly Met 265 270 275 acg gtg gat aca aag tcg aaa ttc aca
gtc gtc act cag ttc atc acc 960Thr Val Asp Thr Lys Ser Lys Phe Thr
Val Val Thr Gln Phe Ile Thr 280 285 290
gac gat ggt acg tcc tcg ggt acc ttg aag gag atc aaa agg ttc tat
1008Asp Asp Gly Thr Ser Ser Gly Thr Leu Lys Glu Ile Lys Arg Phe Tyr
295 300 305 310 gtc cag aac gga aag gtc atc ccg aac tcg gag tcc acg
tgg aca gga 1056Val Gln Asn Gly Lys Val Ile Pro Asn Ser Glu Ser Thr
Trp Thr Gly 315 320 325 gtg tcg ggt aac tcc atc act acg gag tat tgt
aca gcc cag aag tcg 1104Val Ser Gly Asn Ser Ile Thr Thr Glu Tyr Cys
Thr Ala Gln Lys Ser 330 335 340 ctc ttc cag gat cag aac gtc ttc gag
aaa cat gga ggc ttg gaa gga 1152Leu Phe Gln Asp Gln Asn Val Phe Glu
Lys His Gly Gly Leu Glu Gly 345 350 355 atg ggt gcc gca ttg gcc cag
ggt atg gtc ctc gtc atg tcc ttg tgg 1200Met Gly Ala Ala Leu Ala Gln
Gly Met Val Leu Val Met Ser Leu Trp 360 365 370 gac gac cac tcg gcc
aac atg ctc tgg ttg gat tcc aac tac ccc acc 1248Asp Asp His Ser Ala
Asn Met Leu Trp Leu Asp Ser Asn Tyr Pro Thr 375 380 385 390 act gcc
tcg tcc acg aca ccg ggt gtc gca cgc gga act tgt gat atc 1296Thr Ala
Ser Ser Thr Thr Pro Gly Val Ala Arg Gly Thr Cys Asp Ile 395 400 405
tcc tcg gga gtg cct gca gac gtc gag gcg aac cat ccc gac gcc tac
1344Ser Ser Gly Val Pro Ala Asp Val Glu Ala Asn His Pro Asp Ala Tyr
410 415 420 gtg gtc tac tcg aac att aag gtg gga ccc atc ggt tcg aca
ttc aac 1392Val Val Tyr Ser Asn Ile Lys Val Gly Pro Ile Gly Ser Thr
Phe Asn 425 430 435 tcc gga ggc tcg aac cct gga ggc gga acg acc act
act aca acg act 1440Ser Gly Gly Ser Asn Pro Gly Gly Gly Thr Thr Thr
Thr Thr Thr Thr 440 445 450 cag ccg aca aca aca act acc aca gca ggc
aac cct gga ggt aca ggt 1488Gln Pro Thr Thr Thr Thr Thr Thr Ala Gly
Asn Pro Gly Gly Thr Gly 455 460 465 470 gtg gcc cag cac tac gga cag
tgt ggc ggt atc gga tgg aca gga cct 1536Val Ala Gln His Tyr Gly Gln
Cys Gly Gly Ile Gly Trp Thr Gly Pro 475 480 485 act act tgt gca tcg
cct tat acc tgt cag aaa ttg aac gac tac tac 1584Thr Thr Cys Ala Ser
Pro Tyr Thr Cys Gln Lys Leu Asn Asp Tyr Tyr 490 495 500 tcg cag tgt
ttg taa 1599Ser Gln Cys Leu 505 79532PRTAspergillus fumigatus 79Met
Leu Ala Ser Thr Phe Ser Tyr Arg Met Tyr Lys Thr Ala Leu Ile -25 -20
-15 Leu Ala Ala Leu Leu Gly Ser Gly Gln Ala Gln Gln Val Gly Thr Ser
-10 -5 -1 1 5 Gln Ala Glu Val His Pro Ser Met Thr Trp Gln Ser Cys
Thr Ala Gly 10 15 20 Gly Ser Cys Thr Thr Asn Asn Gly Lys Val Val
Ile Asp Ala Asn Trp 25 30 35 Arg Trp Val His Lys Lys Gly Asp Tyr
Thr Asn Cys Tyr Thr Gly Asn 40 45 50 Thr Trp Asp Thr Thr Ile Cys
Pro Asp Asp Ala Thr Cys Ala Ser Asn 55 60 65 70 Cys Ala Leu Glu Gly
Ala Asn Tyr Glu Ser Thr Tyr Gly Val Thr Ala 75 80 85 Ser Gly Asn
Ser Leu Arg Leu Asn Phe Val Thr Thr Ser Gln Gln Lys 90 95 100 Asn
Ile Gly Ser Arg Leu Tyr Met Met Lys Asp Asp Ser Thr Tyr Glu 105 110
115 Met Phe Lys Leu Leu Asn Gln Glu Phe Thr Phe Asp Val Asp Val Ser
120 125 130 Asn Leu Pro Cys Gly Leu Asn Gly Ala Leu Tyr Phe Val Ala
Met Asp 135 140 145 150 Ala Asp Gly Gly Met Ser Lys Tyr Pro Thr Asn
Lys Ala Gly Ala Lys 155 160 165 Tyr Gly Thr Gly Tyr Cys Asp Ser Gln
Cys Pro Arg Asp Leu Lys Phe 170 175 180 Ile Asn Gly Gln Ala Asn Val
Glu Gly Trp Gln Pro Ser Ser Asn Asp 185 190 195 Ala Asn Ala Gly Thr
Gly Asn His Gly Ser Cys Cys Ala Glu Met Asp 200 205 210 Ile Trp Glu
Ala Asn Ser Ile Ser Thr Ala Phe Thr Pro His Pro Cys 215 220 225 230
Asp Thr Pro Gly Gln Val Met Cys Thr Gly Asp Ala Cys Gly Gly Thr 235
240 245 Tyr Ser Ser Asp Arg Tyr Gly Gly Thr Cys Asp Pro Asp Gly Cys
Asp 250 255 260 Phe Asn Ser Phe Arg Gln Gly Asn Lys Thr Phe Tyr Gly
Pro Gly Met 265 270 275 Thr Val Asp Thr Lys Ser Lys Phe Thr Val Val
Thr Gln Phe Ile Thr 280 285 290 Asp Asp Gly Thr Ser Ser Gly Thr Leu
Lys Glu Ile Lys Arg Phe Tyr 295 300 305 310 Val Gln Asn Gly Lys Val
Ile Pro Asn Ser Glu Ser Thr Trp Thr Gly 315 320 325 Val Ser Gly Asn
Ser Ile Thr Thr Glu Tyr Cys Thr Ala Gln Lys Ser 330 335 340 Leu Phe
Gln Asp Gln Asn Val Phe Glu Lys His Gly Gly Leu Glu Gly 345 350 355
Met Gly Ala Ala Leu Ala Gln Gly Met Val Leu Val Met Ser Leu Trp 360
365 370 Asp Asp His Ser Ala Asn Met Leu Trp Leu Asp Ser Asn Tyr Pro
Thr 375 380 385 390 Thr Ala Ser Ser Thr Thr Pro Gly Val Ala Arg Gly
Thr Cys Asp Ile 395 400 405 Ser Ser Gly Val Pro Ala Asp Val Glu Ala
Asn His Pro Asp Ala Tyr 410 415 420 Val Val Tyr Ser Asn Ile Lys Val
Gly Pro Ile Gly Ser Thr Phe Asn 425 430 435 Ser Gly Gly Ser Asn Pro
Gly Gly Gly Thr Thr Thr Thr Thr Thr Thr 440 445 450 Gln Pro Thr Thr
Thr Thr Thr Thr Ala Gly Asn Pro Gly Gly Thr Gly 455 460 465 470 Val
Ala Gln His Tyr Gly Gln Cys Gly Gly Ile Gly Trp Thr Gly Pro 475 480
485 Thr Thr Cys Ala Ser Pro Tyr Thr Cys Gln Lys Leu Asn Asp Tyr Tyr
490 495 500 Ser Gln Cys Leu 505 801599DNAAspergillus
fumigatussig_peptide(1)..(26)CDS(1)..(1596)mat_peptide(79)..(1596)
80atg ttg gcc tcc acg ttc tcc tat cgc atg tac aaa aca gcg ctc atc
48Met Leu Ala Ser Thr Phe Ser Tyr Arg Met Tyr Lys Thr Ala Leu Ile
-25 -20 -15 ttg gca gcc ctc ttg ggc tcg gga cag gca cag cag gtc gga
acc tcg 96Leu Ala Ala Leu Leu Gly Ser Gly Gln Ala Gln Gln Val Gly
Thr Ser -10 -5 -1 1 5 cag gcc gag gtc cat cct tcc atg acg tgg cag
tcg tgt aca gcg ggt 144Gln Ala Glu Val His Pro Ser Met Thr Trp Gln
Ser Cys Thr Ala Gly 10 15 20 ggt tcg tgt acc aca aac aac ggt aaa
gtc gtg atc gat gca aac tgg 192Gly Ser Cys Thr Thr Asn Asn Gly Lys
Val Val Ile Asp Ala Asn Trp 25 30 35 agg tgg gtg cac aag gtc ggc
gac tac acc aac tgt tac aca ggc aac 240Arg Trp Val His Lys Val Gly
Asp Tyr Thr Asn Cys Tyr Thr Gly Asn 40 45 50 aca tgg gat aca acc
atc tgt ccc gac gat gcc act tgt gca tcc aac 288Thr Trp Asp Thr Thr
Ile Cys Pro Asp Asp Ala Thr Cys Ala Ser Asn 55 60 65 70 tgt gca ctc
gag ggt gcc aac tat gag tcg acg tac gga gtg acc gcc 336Cys Ala Leu
Glu Gly Ala Asn Tyr Glu Ser Thr Tyr Gly Val Thr Ala 75 80 85 tcc
gga aac tcg ctc agg ctc aac ttc gtc aca act tcc cag cag aag 384Ser
Gly Asn Ser Leu Arg Leu Asn Phe Val Thr Thr Ser Gln Gln Lys 90 95
100 aac atc ggc tcg cgg ttg tat atg atg aaa gac gat tcc act tac gag
432Asn Ile Gly Ser Arg Leu Tyr Met Met Lys Asp Asp Ser Thr Tyr Glu
105 110 115 atg ttc aag ctc ctc aac cag gaa ttc act ttc gat gtc gac
gtc tcc 480Met Phe Lys Leu Leu Asn Gln Glu Phe Thr Phe Asp Val Asp
Val Ser 120 125 130 aac ctc cct tgt ggc ttg aac gga gcg ctc tac ttc
gtc gcc atg gat 528Asn Leu Pro Cys Gly Leu Asn Gly Ala Leu Tyr Phe
Val Ala Met Asp 135 140 145 150 gcg gat gga ggc atg tcc aag tat cct
acc aac aaa gca gga gcc aag 576Ala Asp Gly Gly Met Ser Lys Tyr Pro
Thr Asn Lys Ala Gly Ala Lys 155 160 165 tat ggt aca ggt tac tgt gat
tcc cag tgt ccc agg gat ctc aag ttc 624Tyr Gly Thr Gly Tyr Cys Asp
Ser Gln Cys Pro Arg Asp Leu Lys Phe 170 175 180 atc aac ggt cag gcc
aac gtc gag ggt tgg cag cct tcg tcg aac gat 672Ile Asn Gly Gln Ala
Asn Val Glu Gly Trp Gln Pro Ser Ser Asn Asp 185 190 195 gcc aac gca
ggt acc ggc aac cac ggt tcc tgt tgt gcc gaa atg gac 720Ala Asn Ala
Gly Thr Gly Asn His Gly Ser Cys Cys Ala Glu Met Asp 200 205 210 att
tgg gaa gcg aac tcg atc tcg acg gcg ttc act cct cac ccg tgt 768Ile
Trp Glu Ala Asn Ser Ile Ser Thr Ala Phe Thr Pro His Pro Cys 215 220
225 230 gat aca ccc gga cag gtg atg tgt aca ggc gac gcc tgt ggc gga
acc 816Asp Thr Pro Gly Gln Val Met Cys Thr Gly Asp Ala Cys Gly Gly
Thr 235 240 245 tac tcg tcg gat cga tat ggc ggt acg tgt gac ccc gac
ggc tgt gac 864Tyr Ser Ser Asp Arg Tyr Gly Gly Thr Cys Asp Pro Asp
Gly Cys Asp 250 255 260 ttc aac tcc ttc agg cag ggc aac aaa aca ttc
tat gga cct ggc atg 912Phe Asn Ser Phe Arg Gln Gly Asn Lys Thr Phe
Tyr Gly Pro Gly Met 265 270 275 acg gtg gat aca aag tcg aaa ttc aca
gtc gtc act cag ttc atc acc 960Thr Val Asp Thr Lys Ser Lys Phe Thr
Val Val Thr Gln Phe Ile Thr 280 285 290 gac gat ggt acg tcc tcg ggt
acc ttg aag gag atc aaa agg ttc tat 1008Asp Asp Gly Thr Ser Ser Gly
Thr Leu Lys Glu Ile Lys Arg Phe Tyr 295 300 305 310 gtc cag aac gga
aag gtc atc ccg aac tcg gag tcc acg tgg aca gga 1056Val Gln Asn Gly
Lys Val Ile Pro Asn Ser Glu Ser Thr Trp Thr Gly 315 320 325 gtg tcg
ggt aac tcc atc act acg gag tat tgt aca gcc cag aag tcg 1104Val Ser
Gly Asn Ser Ile Thr Thr Glu Tyr Cys Thr Ala Gln Lys Ser 330 335 340
ctc ttc cag gat cag aac gtc ttc gag aaa cat gga ggc ttg gaa gga
1152Leu Phe Gln Asp Gln Asn Val Phe Glu Lys His Gly Gly Leu Glu Gly
345 350 355 atg ggt gcc gca ttg gcc cag ggt atg gtc ctc gtc atg tcc
ttg tgg 1200Met Gly Ala Ala Leu Ala Gln Gly Met Val Leu Val Met Ser
Leu Trp 360 365 370 gac gac cac tcg gcc aac atg ctc tgg ttg gat tcc
aac tac ccc acc 1248Asp Asp His Ser Ala Asn Met Leu Trp Leu Asp Ser
Asn Tyr Pro Thr 375 380 385 390 tgg gcc gat cct acg aca ccg ggt gtc
gca cgc gga act tgt gat atc 1296Trp Ala Asp Pro Thr Thr Pro Gly Val
Ala Arg Gly Thr Cys Asp Ile 395 400 405 tcc tcg gga gtg cct gca gac
gtc gag gcg aac cat ccc gac gcc tac 1344Ser Ser Gly Val Pro Ala Asp
Val Glu Ala Asn His Pro Asp Ala Tyr 410 415 420 gtg gtc tac tcg aac
att aag gtg gga ccc atc ggt tcg aca ttc aac 1392Val Val Tyr Ser Asn
Ile Lys Val Gly Pro Ile Gly Ser Thr Phe Asn 425 430 435 tcc gga ggc
tcg aac cct gga ggc gga acg acc act act aca acg act 1440Ser Gly Gly
Ser Asn Pro Gly Gly Gly Thr Thr Thr Thr Thr Thr Thr 440 445 450 cag
ccg aca aca aca act acc aca gca ggc aac cct gga ggt aca ggt 1488Gln
Pro Thr Thr Thr Thr Thr Thr Ala Gly Asn Pro Gly Gly Thr Gly 455 460
465 470 gtg gcc cag cac tac gga cag tgt ggc ggt atc gga tgg aca gga
cct 1536Val Ala Gln His Tyr Gly Gln Cys Gly Gly Ile Gly Trp Thr Gly
Pro 475 480 485 act act tgt gca tcg cct tat acc tgt cag aaa ttg aac
gac tac tac 1584Thr Thr Cys Ala Ser Pro Tyr Thr Cys Gln Lys Leu Asn
Asp Tyr Tyr 490 495 500 tcg cag tgt ttg taa 1599Ser Gln Cys Leu 505
81532PRTAspergillus fumigatus 81Met Leu Ala Ser Thr Phe Ser Tyr Arg
Met Tyr Lys Thr Ala Leu Ile -25 -20 -15 Leu Ala Ala Leu Leu Gly Ser
Gly Gln Ala Gln Gln Val Gly Thr Ser -10 -5 -1 1 5 Gln Ala Glu Val
His Pro Ser Met Thr Trp Gln Ser Cys Thr Ala Gly 10 15 20 Gly Ser
Cys Thr Thr Asn Asn Gly Lys Val Val Ile Asp Ala Asn Trp 25 30 35
Arg Trp Val His Lys Val Gly Asp Tyr Thr Asn Cys Tyr Thr Gly Asn 40
45 50 Thr Trp Asp Thr Thr Ile Cys Pro Asp Asp Ala Thr Cys Ala Ser
Asn 55 60 65 70 Cys Ala Leu Glu Gly Ala Asn Tyr Glu Ser Thr Tyr Gly
Val Thr Ala 75 80 85 Ser Gly Asn Ser Leu Arg Leu Asn Phe Val Thr
Thr Ser Gln Gln Lys 90 95 100 Asn Ile Gly Ser Arg Leu Tyr Met Met
Lys Asp Asp Ser Thr Tyr Glu 105 110 115 Met Phe Lys Leu Leu Asn Gln
Glu Phe Thr Phe Asp Val Asp Val Ser 120 125 130 Asn Leu Pro Cys Gly
Leu Asn Gly Ala Leu Tyr Phe Val Ala Met Asp 135 140 145 150 Ala Asp
Gly Gly Met Ser Lys Tyr Pro Thr Asn Lys Ala Gly Ala Lys 155 160 165
Tyr Gly Thr Gly Tyr Cys Asp Ser Gln Cys Pro Arg Asp Leu Lys Phe 170
175 180 Ile Asn Gly Gln Ala Asn Val Glu Gly Trp Gln Pro Ser Ser Asn
Asp 185 190 195 Ala Asn Ala Gly Thr Gly Asn His Gly Ser Cys Cys Ala
Glu Met Asp 200 205 210 Ile Trp Glu Ala Asn Ser Ile Ser Thr Ala Phe
Thr Pro His Pro Cys 215 220 225 230 Asp Thr Pro Gly Gln Val Met Cys
Thr Gly Asp Ala Cys Gly Gly Thr 235 240 245 Tyr Ser Ser Asp Arg Tyr
Gly Gly Thr Cys Asp Pro Asp Gly Cys Asp 250 255 260 Phe Asn Ser Phe
Arg Gln Gly Asn Lys Thr Phe Tyr Gly Pro Gly Met 265 270 275 Thr Val
Asp Thr Lys Ser Lys Phe Thr Val Val Thr Gln Phe Ile Thr 280 285 290
Asp Asp Gly Thr Ser Ser Gly Thr Leu Lys Glu Ile Lys Arg Phe Tyr 295
300 305 310 Val Gln Asn Gly Lys Val Ile Pro Asn Ser Glu Ser Thr Trp
Thr Gly 315 320 325 Val Ser Gly Asn Ser Ile Thr Thr Glu Tyr Cys Thr
Ala Gln Lys Ser 330 335 340 Leu Phe Gln Asp Gln Asn Val Phe Glu Lys
His Gly Gly Leu Glu Gly 345 350 355 Met Gly Ala Ala Leu Ala Gln Gly
Met Val Leu Val Met Ser Leu Trp 360 365 370 Asp Asp His Ser Ala Asn
Met Leu Trp Leu Asp Ser Asn Tyr Pro Thr 375 380 385 390 Trp Ala Asp
Pro Thr Thr Pro Gly Val Ala Arg Gly Thr Cys Asp Ile 395 400 405 Ser
Ser Gly Val Pro Ala Asp Val Glu
Ala Asn His Pro Asp Ala Tyr 410 415 420 Val Val Tyr Ser Asn Ile Lys
Val Gly Pro Ile Gly Ser Thr Phe Asn 425 430 435 Ser Gly Gly Ser Asn
Pro Gly Gly Gly Thr Thr Thr Thr Thr Thr Thr 440 445 450 Gln Pro Thr
Thr Thr Thr Thr Thr Ala Gly Asn Pro Gly Gly Thr Gly 455 460 465 470
Val Ala Gln His Tyr Gly Gln Cys Gly Gly Ile Gly Trp Thr Gly Pro 475
480 485 Thr Thr Cys Ala Ser Pro Tyr Thr Cys Gln Lys Leu Asn Asp Tyr
Tyr 490 495 500 Ser Gln Cys Leu 505 821599DNAAspergillus
fumigatussig_peptide(1)..(26)CDS(1)..(1596)mat_peptide(79)..(1596)
82atg ttg gcc tcc acg ttc tcc tat cgc atg tac aaa aca gcg ctc atc
48Met Leu Ala Ser Thr Phe Ser Tyr Arg Met Tyr Lys Thr Ala Leu Ile
-25 -20 -15 ttg gca gcc ctc ttg ggc tcg gga cag gca cag cag gtc gga
acc tcg 96Leu Ala Ala Leu Leu Gly Ser Gly Gln Ala Gln Gln Val Gly
Thr Ser -10 -5 -1 1 5 cag gcc gag gtc cat cct tcc atg acg tgg cag
tcg tgt aca gcg ggt 144Gln Ala Glu Val His Pro Ser Met Thr Trp Gln
Ser Cys Thr Ala Gly 10 15 20 ggt tcg tgt acc aca aac aac ggt aaa
gtc gtg atc gat gca aac tgg 192Gly Ser Cys Thr Thr Asn Asn Gly Lys
Val Val Ile Asp Ala Asn Trp 25 30 35 agg tgg gtg cac aag gtc ggc
gac tac acc aac tgt tac aca ggc aac 240Arg Trp Val His Lys Val Gly
Asp Tyr Thr Asn Cys Tyr Thr Gly Asn 40 45 50 aca tgg gat aca acc
atc tgt ccc gac gat gcc act tgt gca tcc aac 288Thr Trp Asp Thr Thr
Ile Cys Pro Asp Asp Ala Thr Cys Ala Ser Asn 55 60 65 70 tgt gca ctc
gag ggt gcc aac tat gag tcg acg tac gga gtg acc gcc 336Cys Ala Leu
Glu Gly Ala Asn Tyr Glu Ser Thr Tyr Gly Val Thr Ala 75 80 85 tcc
gga aac tcg ctc agg ctc aac ttc gtc aca act tcc cag cag aag 384Ser
Gly Asn Ser Leu Arg Leu Asn Phe Val Thr Thr Ser Gln Gln Lys 90 95
100 aac atc ggc tcg cgg ttg tat atg atg aaa gac gat tcc act tac gag
432Asn Ile Gly Ser Arg Leu Tyr Met Met Lys Asp Asp Ser Thr Tyr Glu
105 110 115 atg ttc aag ctc ctc aac cag gaa ttc act ttc gat gtc gac
gtc tcc 480Met Phe Lys Leu Leu Asn Gln Glu Phe Thr Phe Asp Val Asp
Val Ser 120 125 130 aac ctc cct tgt ggc ttg aac gga gcg ctc tac ttc
gtc gcc atg gat 528Asn Leu Pro Cys Gly Leu Asn Gly Ala Leu Tyr Phe
Val Ala Met Asp 135 140 145 150 gcg gat gga ggc atg tcc aag tat cct
acc aac aaa gca gga gcc aag 576Ala Asp Gly Gly Met Ser Lys Tyr Pro
Thr Asn Lys Ala Gly Ala Lys 155 160 165 tat ggt aca ggt tac tgt gat
tcc cag tgt ccc agg gat ctc aag ttc 624Tyr Gly Thr Gly Tyr Cys Asp
Ser Gln Cys Pro Arg Asp Leu Lys Phe 170 175 180 atc aac ggt cag gcc
aac gtc gag ggt tgg cag cct tcg tcg aac gat 672Ile Asn Gly Gln Ala
Asn Val Glu Gly Trp Gln Pro Ser Ser Asn Asp 185 190 195 gcc aac gca
ggt acc ggc aac cac ggt tcc tgt tgt gcc gaa atg gac 720Ala Asn Ala
Gly Thr Gly Asn His Gly Ser Cys Cys Ala Glu Met Asp 200 205 210 att
tgg gaa gcg aac tcg atc tcg acg gcg ttc act cct cac ccg tgt 768Ile
Trp Glu Ala Asn Ser Ile Ser Thr Ala Phe Thr Pro His Pro Cys 215 220
225 230 gat aca ccc gga cag gtg atg tgt aca ggc gac gcc tgt ggc gga
acc 816Asp Thr Pro Gly Gln Val Met Cys Thr Gly Asp Ala Cys Gly Gly
Thr 235 240 245 tac tcg tcg gat cga tat ggc ggt acg tgt gac ccc gac
ggc tgt gac 864Tyr Ser Ser Asp Arg Tyr Gly Gly Thr Cys Asp Pro Asp
Gly Cys Asp 250 255 260 ttc aac tcc ttc agg cag ggc aac aaa aca ttc
tat gga cct ggc atg 912Phe Asn Ser Phe Arg Gln Gly Asn Lys Thr Phe
Tyr Gly Pro Gly Met 265 270 275 acg gtg gat aca aag tcg aaa ttc aca
gtc gtc act cag ttc atc acc 960Thr Val Asp Thr Lys Ser Lys Phe Thr
Val Val Thr Gln Phe Ile Thr 280 285 290 gac gat ggt acg tcc tcg ggt
acc ttg aag gag atc aaa agg ttc tat 1008Asp Asp Gly Thr Ser Ser Gly
Thr Leu Lys Glu Ile Lys Arg Phe Tyr 295 300 305 310 gtc cag aac gga
aag gtc atc ccg aac tcg gag tcc acg tgg aca gga 1056Val Gln Asn Gly
Lys Val Ile Pro Asn Ser Glu Ser Thr Trp Thr Gly 315 320 325 gtg tcg
ggt aac tcc atc act acg gag tat tgt aca gcc cag aag tcg 1104Val Ser
Gly Asn Ser Ile Thr Thr Glu Tyr Cys Thr Ala Gln Lys Ser 330 335 340
ctc ttc cag gat cag aac gtc ttc gag aaa cat gga ggc ttg gaa gga
1152Leu Phe Gln Asp Gln Asn Val Phe Glu Lys His Gly Gly Leu Glu Gly
345 350 355 atg ggt gcc gca ttg gcc cag ggt atg gtc ctc gtc atg tcc
ttg tgg 1200Met Gly Ala Ala Leu Ala Gln Gly Met Val Leu Val Met Ser
Leu Trp 360 365 370 gac gac cac tcg gcc aac atg ctc tgg ttg gat tcc
aac tac ccc acc 1248Asp Asp His Ser Ala Asn Met Leu Trp Leu Asp Ser
Asn Tyr Pro Thr 375 380 385 390 gtc gcc gat cct acg aca ccg ggt gtc
gca cgc gga act tgt gat atc 1296Val Ala Asp Pro Thr Thr Pro Gly Val
Ala Arg Gly Thr Cys Asp Ile 395 400 405 tcc tcg gga gtg cct gca gac
gtc gag gcg aac cat ccc gac gcc tac 1344Ser Ser Gly Val Pro Ala Asp
Val Glu Ala Asn His Pro Asp Ala Tyr 410 415 420 gtg gtc tac tcg aac
att aag gtg gga ccc atc ggt tcg aca ttc aac 1392Val Val Tyr Ser Asn
Ile Lys Val Gly Pro Ile Gly Ser Thr Phe Asn 425 430 435 tcc gga ggc
tcg aac cct gga ggc gga acg acc act act aca acg act 1440Ser Gly Gly
Ser Asn Pro Gly Gly Gly Thr Thr Thr Thr Thr Thr Thr 440 445 450 cag
ccg aca aca aca act acc aca gca ggc aac cct gga ggt aca ggt 1488Gln
Pro Thr Thr Thr Thr Thr Thr Ala Gly Asn Pro Gly Gly Thr Gly 455 460
465 470 gtg gcc cag cac tac gga cag tgt ggc ggt atc gga tgg aca gga
cct 1536Val Ala Gln His Tyr Gly Gln Cys Gly Gly Ile Gly Trp Thr Gly
Pro 475 480 485 act act tgt gca tcg cct tat acc tgt cag aaa ttg aac
gac tac tac 1584Thr Thr Cys Ala Ser Pro Tyr Thr Cys Gln Lys Leu Asn
Asp Tyr Tyr 490 495 500 tcg cag tgt ttg taa 1599Ser Gln Cys Leu 505
83532PRTAspergillus fumigatus 83Met Leu Ala Ser Thr Phe Ser Tyr Arg
Met Tyr Lys Thr Ala Leu Ile -25 -20 -15 Leu Ala Ala Leu Leu Gly Ser
Gly Gln Ala Gln Gln Val Gly Thr Ser -10 -5 -1 1 5 Gln Ala Glu Val
His Pro Ser Met Thr Trp Gln Ser Cys Thr Ala Gly 10 15 20 Gly Ser
Cys Thr Thr Asn Asn Gly Lys Val Val Ile Asp Ala Asn Trp 25 30 35
Arg Trp Val His Lys Val Gly Asp Tyr Thr Asn Cys Tyr Thr Gly Asn 40
45 50 Thr Trp Asp Thr Thr Ile Cys Pro Asp Asp Ala Thr Cys Ala Ser
Asn 55 60 65 70 Cys Ala Leu Glu Gly Ala Asn Tyr Glu Ser Thr Tyr Gly
Val Thr Ala 75 80 85 Ser Gly Asn Ser Leu Arg Leu Asn Phe Val Thr
Thr Ser Gln Gln Lys 90 95 100 Asn Ile Gly Ser Arg Leu Tyr Met Met
Lys Asp Asp Ser Thr Tyr Glu 105 110 115 Met Phe Lys Leu Leu Asn Gln
Glu Phe Thr Phe Asp Val Asp Val Ser 120 125 130 Asn Leu Pro Cys Gly
Leu Asn Gly Ala Leu Tyr Phe Val Ala Met Asp 135 140 145 150 Ala Asp
Gly Gly Met Ser Lys Tyr Pro Thr Asn Lys Ala Gly Ala Lys 155 160 165
Tyr Gly Thr Gly Tyr Cys Asp Ser Gln Cys Pro Arg Asp Leu Lys Phe 170
175 180 Ile Asn Gly Gln Ala Asn Val Glu Gly Trp Gln Pro Ser Ser Asn
Asp 185 190 195 Ala Asn Ala Gly Thr Gly Asn His Gly Ser Cys Cys Ala
Glu Met Asp 200 205 210 Ile Trp Glu Ala Asn Ser Ile Ser Thr Ala Phe
Thr Pro His Pro Cys 215 220 225 230 Asp Thr Pro Gly Gln Val Met Cys
Thr Gly Asp Ala Cys Gly Gly Thr 235 240 245 Tyr Ser Ser Asp Arg Tyr
Gly Gly Thr Cys Asp Pro Asp Gly Cys Asp 250 255 260 Phe Asn Ser Phe
Arg Gln Gly Asn Lys Thr Phe Tyr Gly Pro Gly Met 265 270 275 Thr Val
Asp Thr Lys Ser Lys Phe Thr Val Val Thr Gln Phe Ile Thr 280 285 290
Asp Asp Gly Thr Ser Ser Gly Thr Leu Lys Glu Ile Lys Arg Phe Tyr 295
300 305 310 Val Gln Asn Gly Lys Val Ile Pro Asn Ser Glu Ser Thr Trp
Thr Gly 315 320 325 Val Ser Gly Asn Ser Ile Thr Thr Glu Tyr Cys Thr
Ala Gln Lys Ser 330 335 340 Leu Phe Gln Asp Gln Asn Val Phe Glu Lys
His Gly Gly Leu Glu Gly 345 350 355 Met Gly Ala Ala Leu Ala Gln Gly
Met Val Leu Val Met Ser Leu Trp 360 365 370 Asp Asp His Ser Ala Asn
Met Leu Trp Leu Asp Ser Asn Tyr Pro Thr 375 380 385 390 Val Ala Asp
Pro Thr Thr Pro Gly Val Ala Arg Gly Thr Cys Asp Ile 395 400 405 Ser
Ser Gly Val Pro Ala Asp Val Glu Ala Asn His Pro Asp Ala Tyr 410 415
420 Val Val Tyr Ser Asn Ile Lys Val Gly Pro Ile Gly Ser Thr Phe Asn
425 430 435 Ser Gly Gly Ser Asn Pro Gly Gly Gly Thr Thr Thr Thr Thr
Thr Thr 440 445 450 Gln Pro Thr Thr Thr Thr Thr Thr Ala Gly Asn Pro
Gly Gly Thr Gly 455 460 465 470 Val Ala Gln His Tyr Gly Gln Cys Gly
Gly Ile Gly Trp Thr Gly Pro 475 480 485 Thr Thr Cys Ala Ser Pro Tyr
Thr Cys Gln Lys Leu Asn Asp Tyr Tyr 490 495 500 Ser Gln Cys Leu 505
841599DNAAspergillus
fumigatussig_peptide(1)..(26)CDS(1)..(1596)mat_peptide(79)..(1596)
84atg ttg gcc tcc acg ttc tcc tat cgc atg tac aaa aca gcg ctc atc
48Met Leu Ala Ser Thr Phe Ser Tyr Arg Met Tyr Lys Thr Ala Leu Ile
-25 -20 -15 ttg gca gcc ctc ttg ggc tcg gga cag gca cag cag gtc gga
acc tcg 96Leu Ala Ala Leu Leu Gly Ser Gly Gln Ala Gln Gln Val Gly
Thr Ser -10 -5 -1 1 5 cag gcc gag gtc cat cct tcc atg acg tgg cag
tcg tgt aca gcg ggt 144Gln Ala Glu Val His Pro Ser Met Thr Trp Gln
Ser Cys Thr Ala Gly 10 15 20 ggt tcg tgt acc aca aac aac ggt aaa
gtc gtg atc gat gca aac tgg 192Gly Ser Cys Thr Thr Asn Asn Gly Lys
Val Val Ile Asp Ala Asn Trp 25 30 35 agg tgg gtg cac aag gtc ggc
gac tac acc aac tgt tac aca ggc aac 240Arg Trp Val His Lys Val Gly
Asp Tyr Thr Asn Cys Tyr Thr Gly Asn 40 45 50 aca tgg gat aca acc
atc tgt ccc gac gat gcc act tgt gca tcc aac 288Thr Trp Asp Thr Thr
Ile Cys Pro Asp Asp Ala Thr Cys Ala Ser Asn 55 60 65 70 tgt gca ctc
gag ggt gcc aac tat gag tcg acg tac gga gtg acc gcc 336Cys Ala Leu
Glu Gly Ala Asn Tyr Glu Ser Thr Tyr Gly Val Thr Ala 75 80 85 tcc
gga aac tcg ctc agg ctc aac ttc gtc aca act tcc cag cag aag 384Ser
Gly Asn Ser Leu Arg Leu Asn Phe Val Thr Thr Ser Gln Gln Lys 90 95
100 aac atc ggc tcg cgg ttg tat atg atg aaa gac gat tcc act tac gag
432Asn Ile Gly Ser Arg Leu Tyr Met Met Lys Asp Asp Ser Thr Tyr Glu
105 110 115 atg ttc aag ctc ctc aac cag gaa ttc act ttc gat gtc gac
gtc tcc 480Met Phe Lys Leu Leu Asn Gln Glu Phe Thr Phe Asp Val Asp
Val Ser 120 125 130 aac ctc cct tgt ggc ttg aac gga gcg ctc tac ttc
gtc gcc atg gat 528Asn Leu Pro Cys Gly Leu Asn Gly Ala Leu Tyr Phe
Val Ala Met Asp 135 140 145 150 gcg gat gga ggc atg tcc aag tat cct
acc aac aaa gca gga gcc aag 576Ala Asp Gly Gly Met Ser Lys Tyr Pro
Thr Asn Lys Ala Gly Ala Lys 155 160 165 tat ggt aca ggt tac tgt gat
tcc cag tgt ccc agg gat ctc aag ttc 624Tyr Gly Thr Gly Tyr Cys Asp
Ser Gln Cys Pro Arg Asp Leu Lys Phe 170 175 180 atc aac ggt cag gcc
aac gtc gag ggt tgg cag cct tcg tcg aac gat 672Ile Asn Gly Gln Ala
Asn Val Glu Gly Trp Gln Pro Ser Ser Asn Asp 185 190 195 gcc aac gca
ggt acc ggc aac cac ggt tcc tgt tgt gcc gaa atg gac 720Ala Asn Ala
Gly Thr Gly Asn His Gly Ser Cys Cys Ala Glu Met Asp 200 205 210 att
tgg gaa gcg aac tcg atc tcg acg gcg ttc act cct cac ccg tgt 768Ile
Trp Glu Ala Asn Ser Ile Ser Thr Ala Phe Thr Pro His Pro Cys 215 220
225 230 gat aca ccc gga cag gtg atg tgt aca ggc gac gcc tgt ggc gga
acc 816Asp Thr Pro Gly Gln Val Met Cys Thr Gly Asp Ala Cys Gly Gly
Thr 235 240 245 tac tcg tcg gat cga tat ggc ggt acg tgt gac ccc gac
ggc tgt gac 864Tyr Ser Ser Asp Arg Tyr Gly Gly Thr Cys Asp Pro Asp
Gly Cys Asp 250 255 260 ttc aac tcc ttc agg cag ggc aac aaa aca ttc
tat gga cct ggc atg 912Phe Asn Ser Phe Arg Gln Gly Asn Lys Thr Phe
Tyr Gly Pro Gly Met 265 270 275 acg gtg gat aca aag tcg aaa ttc aca
gtc gtc act cag ttc atc acc 960Thr Val Asp Thr Lys Ser Lys Phe Thr
Val Val Thr Gln Phe Ile Thr 280 285 290 gac gat ggt acg tcc tcg ggt
acc ttg aag gag atc aaa agg ttc tat 1008Asp Asp Gly Thr Ser Ser Gly
Thr Leu Lys Glu Ile Lys Arg Phe Tyr 295 300 305 310 gtc cag aac gga
aag gtc atc ccg aac tcg gag tcc acg tgg aca gga 1056Val Gln Asn Gly
Lys Val Ile Pro Asn Ser Glu Ser Thr Trp Thr Gly 315 320 325 gtg tcg
ggt aac tcc atc act acg gag tat tgt aca gcc cag aag tcg 1104Val Ser
Gly Asn Ser Ile Thr Thr Glu Tyr Cys Thr Ala Gln Lys Ser 330 335 340
ctc ttc cag gat cag aac gtc ttc gag aaa cat gga ggc ttg gaa gga
1152Leu Phe Gln Asp Gln Asn Val Phe Glu Lys His Gly Gly Leu Glu Gly
345 350 355 atg ggt gcc gca ttg gcc cag ggt atg gtc ctc gtc atg tcc
ttg tgg 1200Met Gly Ala Ala Leu Ala Gln Gly Met Val Leu Val Met Ser
Leu Trp 360 365 370 gac gac cac tcg gcc aac atg ctc tgg ttg gat tcc
aac tac ccc acc 1248Asp Asp His Ser Ala Asn Met Leu Trp Leu Asp Ser
Asn Tyr Pro Thr 375 380 385 390 aac gcc gat cct acg aca ccg ggt gtc
gca cgc gga act tgt gat atc
1296Asn Ala Asp Pro Thr Thr Pro Gly Val Ala Arg Gly Thr Cys Asp Ile
395 400 405 tcc tcg gga gtg cct gca gac gtc gag gcg aac cat ccc gac
gcc tac 1344Ser Ser Gly Val Pro Ala Asp Val Glu Ala Asn His Pro Asp
Ala Tyr 410 415 420 gtg gtc tac tcg aac att aag gtg gga ccc atc ggt
tcg aca ttc aac 1392Val Val Tyr Ser Asn Ile Lys Val Gly Pro Ile Gly
Ser Thr Phe Asn 425 430 435 tcc gga ggc tcg aac cct gga ggc gga acg
acc act act aca acg act 1440Ser Gly Gly Ser Asn Pro Gly Gly Gly Thr
Thr Thr Thr Thr Thr Thr 440 445 450 cag ccg aca aca aca act acc aca
gca ggc aac cct gga ggt aca ggt 1488Gln Pro Thr Thr Thr Thr Thr Thr
Ala Gly Asn Pro Gly Gly Thr Gly 455 460 465 470 gtg gcc cag cac tac
gga cag tgt ggc ggt atc gga tgg aca gga cct 1536Val Ala Gln His Tyr
Gly Gln Cys Gly Gly Ile Gly Trp Thr Gly Pro 475 480 485 act act tgt
gca tcg cct tat acc tgt cag aaa ttg aac gac tac tac 1584Thr Thr Cys
Ala Ser Pro Tyr Thr Cys Gln Lys Leu Asn Asp Tyr Tyr 490 495 500 tcg
cag tgt ttg taa 1599Ser Gln Cys Leu 505 85532PRTAspergillus
fumigatus 85Met Leu Ala Ser Thr Phe Ser Tyr Arg Met Tyr Lys Thr Ala
Leu Ile -25 -20 -15 Leu Ala Ala Leu Leu Gly Ser Gly Gln Ala Gln Gln
Val Gly Thr Ser -10 -5 -1 1 5 Gln Ala Glu Val His Pro Ser Met Thr
Trp Gln Ser Cys Thr Ala Gly 10 15 20 Gly Ser Cys Thr Thr Asn Asn
Gly Lys Val Val Ile Asp Ala Asn Trp 25 30 35 Arg Trp Val His Lys
Val Gly Asp Tyr Thr Asn Cys Tyr Thr Gly Asn 40 45 50 Thr Trp Asp
Thr Thr Ile Cys Pro Asp Asp Ala Thr Cys Ala Ser Asn 55 60 65 70 Cys
Ala Leu Glu Gly Ala Asn Tyr Glu Ser Thr Tyr Gly Val Thr Ala 75 80
85 Ser Gly Asn Ser Leu Arg Leu Asn Phe Val Thr Thr Ser Gln Gln Lys
90 95 100 Asn Ile Gly Ser Arg Leu Tyr Met Met Lys Asp Asp Ser Thr
Tyr Glu 105 110 115 Met Phe Lys Leu Leu Asn Gln Glu Phe Thr Phe Asp
Val Asp Val Ser 120 125 130 Asn Leu Pro Cys Gly Leu Asn Gly Ala Leu
Tyr Phe Val Ala Met Asp 135 140 145 150 Ala Asp Gly Gly Met Ser Lys
Tyr Pro Thr Asn Lys Ala Gly Ala Lys 155 160 165 Tyr Gly Thr Gly Tyr
Cys Asp Ser Gln Cys Pro Arg Asp Leu Lys Phe 170 175 180 Ile Asn Gly
Gln Ala Asn Val Glu Gly Trp Gln Pro Ser Ser Asn Asp 185 190 195 Ala
Asn Ala Gly Thr Gly Asn His Gly Ser Cys Cys Ala Glu Met Asp 200 205
210 Ile Trp Glu Ala Asn Ser Ile Ser Thr Ala Phe Thr Pro His Pro Cys
215 220 225 230 Asp Thr Pro Gly Gln Val Met Cys Thr Gly Asp Ala Cys
Gly Gly Thr 235 240 245 Tyr Ser Ser Asp Arg Tyr Gly Gly Thr Cys Asp
Pro Asp Gly Cys Asp 250 255 260 Phe Asn Ser Phe Arg Gln Gly Asn Lys
Thr Phe Tyr Gly Pro Gly Met 265 270 275 Thr Val Asp Thr Lys Ser Lys
Phe Thr Val Val Thr Gln Phe Ile Thr 280 285 290 Asp Asp Gly Thr Ser
Ser Gly Thr Leu Lys Glu Ile Lys Arg Phe Tyr 295 300 305 310 Val Gln
Asn Gly Lys Val Ile Pro Asn Ser Glu Ser Thr Trp Thr Gly 315 320 325
Val Ser Gly Asn Ser Ile Thr Thr Glu Tyr Cys Thr Ala Gln Lys Ser 330
335 340 Leu Phe Gln Asp Gln Asn Val Phe Glu Lys His Gly Gly Leu Glu
Gly 345 350 355 Met Gly Ala Ala Leu Ala Gln Gly Met Val Leu Val Met
Ser Leu Trp 360 365 370 Asp Asp His Ser Ala Asn Met Leu Trp Leu Asp
Ser Asn Tyr Pro Thr 375 380 385 390 Asn Ala Asp Pro Thr Thr Pro Gly
Val Ala Arg Gly Thr Cys Asp Ile 395 400 405 Ser Ser Gly Val Pro Ala
Asp Val Glu Ala Asn His Pro Asp Ala Tyr 410 415 420 Val Val Tyr Ser
Asn Ile Lys Val Gly Pro Ile Gly Ser Thr Phe Asn 425 430 435 Ser Gly
Gly Ser Asn Pro Gly Gly Gly Thr Thr Thr Thr Thr Thr Thr 440 445 450
Gln Pro Thr Thr Thr Thr Thr Thr Ala Gly Asn Pro Gly Gly Thr Gly 455
460 465 470 Val Ala Gln His Tyr Gly Gln Cys Gly Gly Ile Gly Trp Thr
Gly Pro 475 480 485 Thr Thr Cys Ala Ser Pro Tyr Thr Cys Gln Lys Leu
Asn Asp Tyr Tyr 490 495 500 Ser Gln Cys Leu 505
* * * * *